The authors thank previous and current members of the laboratory for development of the protocols. The work was supported by funding from the Canadian Institutes of Health Research (CIHR) [grant # MOP-130527] and from the Natural Sciences and Engineering Research Council of Canada [NSERC-355306-2012]. NG is recipient of a Tier II Canada Research Chair. AR holds a studentship from the training program of the Respiratory Health Research Network from the Fonds de la recherche du Qu&#233;bec-Sant&#233; (FRQS).

NOTE: The protocol is described here using A549 cells infected with Sendai virus (SeV). However, the protocol for SDS-PAGE and native PAGE also works with all human and murine cell types tested so far, particularly myeloid cells stimulated with various IRF3-activating stimuli 9,15,19,24,25.
1. Infection of A549 Cells
<ol>
	<li>Maintain A549 cells in culture in a 15 cm plate at 37 &#176;C/5% CO2 in 20 ml F12K/Ham medium containing 10% heat-inactivated fetal bovine serum (HI-FBS) and 1% L-glutamine (complete F12K/Ham medium). 
	NOTE: All the solutions used for cell culture and treatments must be sterile.</li>
	<li>At 24 hr before the infection, aspirate the medium and wash the cells with 10 ml of distilled phosphate buffered saline (D-PBS) at RT.</li>
	<li>Add 1 ml of 0.25% trypsin-EDTA solution to each plate to cover the cells and incubate at 37 &#176;C for 3 min.</li>
	<li>Stop the incubation as soon as the cells start to detach from the plate by softly tapping the plate with one hand. Inactivate the trypsin by adding 10 ml of pre-heated complete F12K/Ham medium per plate and transfer the cells to a 15 ml conical tube.</li>
	<li>Centrifuge at 350 x g for 3 min at RT. Remove the supernatant and resuspend the cell pellet in 8 ml of complete F12K/Ham medium to obtain a homogeneous single cell suspension.</li>
	<li>Count the cells using a hemocytometer. Seed the cells at a density of 1 x 106 cells per 60 mm plate in 4 ml of pre-heated complete F12K/Ham medium. Incubate for 20 - 24 hr at 37 &#176;C/5% CO2. After 20 - 24 hr, the cells form a 90% confluent monolayer (this corresponds to 1.5 x 106 cells).</li>
	<li>Remove the medium and wash the cells with 2 ml of serum-free F12K/Ham medium (SFM). Add 2 ml of fresh SFM per 60 mm plate.</li>
	<li>Thaw an aliquot of Sendai virus (SeV) (stored aliquoted at -80 &#176;C) on ice and vortex briefly.</li>
	<li>Dilute the virus in pre-heated SFM to obtain 60 HAU/100 &#181;l. Mix by pipetting up and down softly and add 100 &#181;l of diluted virus per plate to perform the infection at 40 HAU/106 cells. Do not add virus in the non-infected control plate.</li>
	<li>Incubate the cells in the incubator at 37 &#176;C/5% CO2. Agitate the plates by hand 3 or 4 times, or using an automatic orbital or rocking shaker placed directly in the incubator, during the first hour of infection.</li>
	<li>At 2 hr post-infection, add 2 ml of F12K/Ham medium containing 20% HI-FBS to obtain a final concentration of 10% HI-FBS.</li>
	<li>Incubate the cells in the incubator at 37 &#176;C/5% CO2 for an additional 1, 4 and 7 hr to reach total infection times of 3, 6, and 9 hr, respectively. At each of these time points, proceed to step 2.1.</li>
</ol>
2. Preparation of Whole Cell Extracts (WCE)
<ol>
	<li>Remove the infection medium. Harvest the cells by scraping in 1 ml of ice-cold D-PBS and transfer the cell suspension to a pre-chilled 1.5 ml centrifuge tube.</li>
	<li>Pellet the cells by centrifugation at 16,000 x g at 4 &#176;C for 20 sec and carefully decant all traces of D-PBS. 
	NOTE: At this step, the cell pellet can be directly subjected to protein extraction or flash-frozen in liquid nitrogen or dry ice/ethanol bath and stored at -80 &#176;C until lysis.</li>
	<li>Prepare the lysis buffer containing 50 mM HEPES pH 7.4, 150 mM NaCl, 5 mM EDTA, 10% glycerol and 1% Nonidet P-40 in deionized water (ddH2O). Extemporarily add protease (1 &#181;g/ml leupeptin and 2 &#181;g/ml aprotinin) and phosphatase inhibitors (5 mM sodium fluoride, 1 mM activated sodium orthovanadate, 2 mM p-nitrophenyl phosphate and 10 mM &#946;-glycerophosphate pH 7.5). 
	NOTE: The lysis buffer without inhibitors can be stored at 4 &#176;C. A specific protocol for the activation of sodium orthovanadate is described in the Table of Specific Reagents/Equipment.</li>
	<li>Resuspend the cell pellet in 70 &#181;l of lysis buffer. Typically the lysate concentration will be around 2 &#181;g/&#181;l.</li>
	<li>Incubate on ice for 20 min. Flash-freeze the lysate by incubation in a liquid nitrogen bath for 15 sec. Thaw the lysate at RT until it is completely melted and vortex for 10 sec. Repeat the freeze/thaw/vortex cycle 3 times.
	<ol>
		<li>Alternatively, perform the freezing step in an ethanol/dry ice bath for 1 min.</li>
	</ol>
	</li>
	<li>Centrifuge at 16,000 x g at 4 &#176;C for 20 min. Transfer the supernatant (corresponding to the WCE) to a new pre-chilled 1.5 ml centrifuge tube. Keep the WCE on ice at all times.</li>
	<li>Quantify proteins using any protein quantification procedure compatible with the lysis buffer such as Bradford-based protein assay26.</li>
</ol>
3. Resolution of WCE by High Resolution SDS-PAGE
<ol>
	<li>Prepare three denaturing electrophoresis gels.
	<ol>
		<li>For the detection of IRF3 forms, pour two gels of a minimum of 16 cm length with a separation gel composed of 7.5% acrylamide/bis-acrylamide (37.5:1), 375 mM Tris-HCl pH 8.8 (RT), 0.1% sodium dodecyl sulfate (SDS), 1% ammonium persulfate and 0.1% TEMED in ddH2O and a stacking gel composed of 4% acrylamide, 62.5 mM Tris-HCl pH 6.8 (RT), 0.05% SDS, 1% ammonium persulfate and 0.1% TEMED in ddH2O. 
		Caution! Acrylamide, TEMED and SDS are toxic and/or irritant. Wear protective gloves and manipulate under a fume hood.</li>
		<li>For the detection of SeV proteins, pour one gel of a minimum of 8.5 cm length with characteristics similar to the gel described in 3.1.1, except that the separation gel contains 12% acrylamide.</li>
	</ol>
	</li>
	<li>Denature the WCE obtained in step 2.6 by adding 1:4 (v/v) 5x loading buffer (125 mM Tris-HCl pH 6.8 (RT), 10% SDS, 20% (p/v) glycerol, 0.0005% bromophenol blue and 25% &#946;-mercaptoethanol in ddH2O) followed by heating at 100 &#176;C for 10 min. Quick spin the tubes to bring down the condensation that forms in the cap. 
	Caution! &#946;-mercaptoethanol is toxic by inhalation. Wear protective gloves and manipulate under a fume hood.</li>
	<li>Mount the gels in the migration apparatus. Fill the upper and lower chambers with running buffer containing 25 mM Tris-Base, 0.1% SDS and 192 mM glycine in distilled water (dH2O).</li>
	<li>Load 14 &#181;l of molecular weight standard in one well of each 16 cm gel and 7 &#181;l of molecular weight standard in one well of the 8.5 cm gel. Load 30 &#181;g of denatured WCE (prepared as described in step 3.2) per well of the two gels prepared in step 3.1.1 for IRF3 forms detection. Load 8 - 10 &#181;g of denatured WCE (prepared as described in step 3.2) per well on the gel prepared in step 3.1.2 for SeV analysis.</li>
	<li>Run the gels at 30 mA constant current until the migration front reaches the bottom of the gel. 
	NOTE: Migration typically lasts approximately 3 hr for a 16 cm gel and 45 min for an 8.5 cm gel.</li>
	<li>Proceed to the transfer onto nitrocellulose membranes (step 5).</li>
</ol>
4. Analysis of IRF3 Dimerization by Native-PAGE
NOTE: This method was originally described by the group of Dr. T. Fujita27.
<ol>
	<li>Prepare the upper (-) and lower (+) chamber electrophoresis buffers. The upper chamber buffer consists of 25 mM Tris-HCl pH 8.4 (RT), 192 mM glycine and 1% sodium deoxycholate (DOC) in ddH2O. The lower chamber buffer contains 25 mM Tris-HCl pH 8.4 (RT) and 192 mM glycine in ddH2O. 
	NOTE: The upper and lower chamber electrophoresis buffers can be stored at 4 &#176;C until use. However, make sure that they are pre-warmed to RT before performing the electrophoresis.</li>
	<li>Pour a non-denaturing resolving gel of a minimum of 8.5 cm length containing 7.5% acrylamide/bis-acrylamide (37.5:1), 375 mM Tris-HCl pH 8.8 (RT), 1% ammonium persulfate and 0.1% TEMED in ddH2O.</li>
	<li>Pre-run the gel at 40 mA constant current for 30 min on ice. Press the running apparatus into the ice approximately to the level of the lower chamber electrophoresis buffer. It is important that the gel is not in the ice.</li>
	<li>During pre-run, mix WCE kept on ice with 2x native-PAGE loading buffer 1:1 (v/v) containing 125 mM Tris-HCl pH 6.8 (RT), 30% glycerol and 0.1% bromophenol blue in ddH2O.</li>
	<li>Load 8 - 10 &#181;g WCE (prepared as described in step 4.4) immediately at the end of the pre-run.</li>
	<li>Run the gel at 25 mA constant current on ice, as described above for the pre-run, until the migration front reaches the bottom of the gel (approximately 40 min).</li>
</ol>
5. Immunoblot Analysis of IRF3 Species
<ol>
	<li>Prepare the transfer buffer containing 25 mM Tris-Base and 192 mM glycine in ddH2O. Refrigerate the transfer buffer at 4 &#176;C before use.</li>
	<li>Wet three pieces of nitrocellulose membrane cut to a size slightly larger than the gels in a plastic/glass box containing the transfer buffer. Indicate the orientation of the membrane by cutting one corner. The transfer buffer can be reused 3 times. Store the buffer at 4 &#176;C between uses.</li>
	<li>Uncast the gels (SDS-PAGE and native-PAGE) and cut one corner of each gel for proper orientation.
	<ol>
		<li>For native-PAGE, incubate the gel at RT with gentle agitation for at least 30 min in the SDS-PAGE running buffer to remove the DOC before transferring to the transfer buffer.</li>
	</ol>
	</li>
	<li>Incubate the gels (SDS-PAGE and native-PAGE) in the transfer buffer for 5 - 10 min.</li>
	<li>Mount a transfer sandwich per gel in a transfer cassette with the membrane towards the positive electrode (foam pad/filter paper/membrane/gel/filter paper/foam pad). Be careful to remove all the bubbles in between the layers of the sandwich.</li>
	<li>Perform the transfer as recommended by the manufacturer for the transfer apparatus used. 
	NOTE: Perform all incubations and washes in the next steps on a rocking or orbital shaking platform.</li>
	<li>At the end of the transfer time, incubate the membranes for 15 min in the fixation solution containing 7% acetic acid, 40% ethanol and 3% glycerol in ddH2O. Wash the membranes 3x 5 min in PBS (137 mM NaCl, 2.7 mM KCl, 10.2 mM Na2HPO4 and 1.8 mM KH2PO4 in dH2O). 
	Caution! Acetic acid is toxic, irritant and flammable. Wear protective gloves and manipulate under the fume hood. 
	NOTE: The fixation solution can be reused multiple times.</li>
	<li>For the native-PAGE membrane proceed directly to step 5.11.</li>
	<li>Rinse the three SDS-PAGE membranes quickly in dH2O before incubating them for 1 min in red ponceau solution containing 6.57 mM red ponceau and 1% acetic acid in ddH2O. 
	NOTE: The red ponceau solution can be reused several times.</li>
	<li>Rinse the membranes in dH2O until the background is white enough to see the protein bands stained in red. Note the markers with a pencil and cut the excess membrane around the proteins. Destain the membranes by incubation for 5 min in PBS under agitation.</li>
	<li>Incubate the membranes for 1 hr at RT or O/N&#160;at 4 &#176;C in the blocking solution (PBS containing 0.05% Tween 20 and 5% non-fat dry milk (PBS-T-milk). Wash the membranes 3x 5 min in PBS-T. 
	NOTE: The PBS-T wash is optional and only strictly required when applying antibodies diluted in PBS-T containing 5% bovine serum albumin (PBS-T-BSA) (Figure 1 and Table 1) in the next step.</li>
	<li>Incubate the four membranes (from SDS-PAGE and native-PAGE) with the primary antibodies according to the sequential order detailed in Figure 1 and Table 1. Perform 5x 5 min washes in PBS-T.</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/53723/53723fig1.jpg" /> 
Figure 1. Sequence of Immunoblot Analyses. The schematic describes the individual SDS-PAGE and native-PAGE gels required to detect the various IRF3 phosphorylated and monomeric/dimeric forms. The specific order of antibodies applied to the membrane resulting from each gel in the immunoblot procedure is described. Note that the anti-actin antibodies are used first to ensure equal loading of the samples before applying any other specific antibodies. The alternate sequence, with anti-actin antibodies being applied after the anti-phospho-IRF3 antibodies, also works. Stripping is used between anti-actin and anti-SeV antibodies, or between anti-phospho-IRF3 and anti-IRF3 antibodies because of overlapping size of the signals. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53723/53723fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Primary antibodies</td>
			<td>Dilution</td>
			<td>Dilution buffer</td>
			<td>Incubation</td>
			<td>Secondary antibodies</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Anti-Actin</td>
			<td>1/10,000</td>
			<td>PBS-T-BSA</td>
			<td>15 min at RT</td>
			<td>Anti-mouse</td>
			<td>Use after SDS-PAGE&#160; 
			Diluted antibodyies can be reused several times if stored at 4 &#176;C in the presence of 0.02 % Sodium Azide.</td>
		</tr>
		<tr>
			<td>Anti-IRF3-P-Ser386</td>
			<td>1/200</td>
			<td>PBS-T-BSA</td>
			<td>O/N at 4 &#176;C</td>
			<td>Anti-rabbit</td>
			<td>Use after Native-PAGE.&#160; 
			It is not recommended to reuse the diluted antibody&#160;</td>
		</tr>
		<tr>
			<td>Anti-IRF3-P-Ser396</td>
			<td>1/10,000</td>
			<td>PBS-T-BSA</td>
			<td>O/N at 4 &#176;C</td>
			<td>Anti-rabbit</td>
			<td>Use after SDS-PAGE.&#160; 
			It is not recommended to reuse the diluted antibody Anti-IRF3-P-396 is also available from Cell Signaling. Optimal dilution was defined as 1/1,000 for this antibody, but may vary between lots.&#160;&#160;</td>
		</tr>
		<tr>
			<td>Anti-IRF3-P-Ser398</td>
			<td>1/10,000</td>
			<td>PBS-T-BSA</td>
			<td>O/N at 4 &#176;C</td>
			<td>Anti-rabbit</td>
			<td>Use after SDS-PAGE.&#160; It is not recommended to reuse the diluted antibody&#160;</td>
		</tr>
		<tr>
			<td>Anti-IRF3 full length</td>
			<td>1/7,500</td>
			<td>PBS-T-milk</td>
			<td>3 hr at RT</td>
			<td>Anti-rabbit</td>
			<td>Use after SDS-PAGE.&#160;&#160; Anti-IRF3 full length antibody can be used after native-PAGE, but it is less sensitive to detect the monomer. Diluted antibodies can be reused several times if stored at 4 &#176;C in the presence of 0.02 % Sodium Azide.&#160;&#160;</td>
		</tr>
		<tr>
			<td>Anti-IRF3-NES</td>
			<td>0.5&#160; &#956;g/ml</td>
			<td>PBS-T-milk</td>
			<td>3 hr at RT</td>
			<td>Anti-rabbit</td>
			<td>Use after Native-PAGE. Diluted antibodyies can be reused several times if stored at 4 &#176;C in the presence of 0.02 % Sodium Azide.</td>
		</tr>
		<tr>
			<td>Anti-SeV</td>
			<td>1/14,000</td>
			<td>PBS-T-BSA</td>
			<td>3 hr at RT</td>
			<td>Anti-rabbit</td>
			<td>Use after SDS-PAGE. Diluted antibodyies can be reused several times if stored at 4 &#176;C in the presence of 0.02 % Sodium Azide.</td>
		</tr>
		<tr>
			<td colspan="6">NOTE: Dilution and buffer used for HRP-coupled secondary antibodies need to be optimized as it varies from one company to the other.</td>
		</tr>
	</tbody>
</table>
Table 1. Specifications of Antibodies used in the Immunoblotting Procedure.
<ol start="13">
	<li>Incubate the membranes with the horseradish peroxidase (HRP)-conjugated secondary antibodies as detailed in Figure 1 and Table 1. Wash the membranes 5x&#160;5 min in PBS-T, followed by 2x&#160;5 min in PBS to fully remove traces of Tween.</li>
	<li>Incubate the membranes for 1 min in a volume of enhanced chemiluminescence reagent sufficient to fully cover the membranes. Dry the membranes using filter paper.</li>
	<li>Place the membranes in a luminescent image analyzer to visualize the immunoreactive bands.
	<ol>
		<li>Alternatively, perform the detection of immunoreactive bands using sensitive X-Ray films.</li>
	</ol>
	</li>
	<li>Wash the membranes 3x 5 min in PBS. 
	NOTE: At this step the membranes can be kept dry. However, if further stripping is required, it is better to perform the stripping before drying the membrane. Membranes can also be stored for short-term in PBS.</li>
	<li>For the membranes that do not require stripping between incubation with antibodies (see Figure 1) proceed directly to step 5.20.</li>
	<li>When stripping is required between antibodies (see Figure 1), incubate the membranes in pre-warmed stripping solution containing 2% SDS, 62.5 mM Tris-HCl pH 6.8 (RT) and 0.7% &#946;-mercaptoethanol in ddH2O at 50 &#176;C for 20 min under regular agitation. Wash the membranes 3x 5 min in PBS. 
	NOTE: Agitation during the stripping procedure is key. Stripping can be performed in a hybridization oven. Alternatively, stripping can be performed using membranes sealed in plastic bags and immersed in a water bath. In this case, it is important to agitate the membranes 4 - 5 times during the incubation.</li>
	<li>Incubate the membranes for 1 hr at RT or O/N at 4 &#176;C in PBS-T-milk.</li>
	<li>Repeat steps 5.12 to 5.16 according to Figure 1.</li>
</ol>

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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+the+minimal+phosphoacceptor+site+required+for+in+vivo+activation+of+interferon+regulatory+factor+3+in+response+to+virus+and+double-stranded+RNA.">Identification of the minimal phosphoacceptor site required for in vivo activation of interferon regulatory factor 3 in response to virus and double-stranded RNA.</a> J Biol Chem. 278 (11), 9441-9447 (2003).</li><li>Bergstroem, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+novel+in+vivo+virus-targeted+phosphorylation+site+in+interferon+regulatory+factor-3+(IRF3).">Identification of a novel in vivo virus-targeted phosphorylation site in interferon regulatory factor-3 (IRF3).</a> J Biol Chem. 285 (32), 24904-24914 (2010).</li><li>Takahasi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ser386+phosphorylation+of+transcription+factor+IRF-3+induces+dimerization+and+association+with+CBP/p300+without+overall+conformational+change.">Ser386 phosphorylation of transcription factor IRF-3 induces dimerization and association with CBP/p300 without overall conformational change.</a> Genes Cells. 15 (8), 901-910 (2010).</li><li>Noyce, R. S., Collins, S. E., Mossman, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+modification+of+interferon+regulatory+factor+3+following+virus+particle+entry.">Differential modification of interferon regulatory factor 3 following virus particle entry.</a> J Virol. 83 (9), 4013-4022 (2009).</li><li>McCoy, C. E., Carpenter, S., Palsson-McDermott, E. M., Gearing, L. J., O'Neill, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucocorticoids+inhibit+IRF3+phosphorylation+in+response+to+Toll-like+receptor-3+and+-4+by+targeting+TBK1+activation.">Glucocorticoids inhibit IRF3 phosphorylation in response to Toll-like receptor-3 and -4 by targeting TBK1 activation.</a> J Biol Chem. 283 (21), 14277-14285 (2008).</li><li>Oliere, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HTLV-1+evades+type+I+interferon+antiviral+signaling+by+inducing+the+suppressor+of+cytokine+signaling+1+(SOCS1).">HTLV-1 evades type I interferon antiviral signaling by inducing the suppressor of cytokine signaling 1 (SOCS1).</a> PLoS Pathog. 6 (11), e1001177 (2010).</li><li>Kato, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+type-specific+involvement+of+RIG-I+in+antiviral+response.">Cell type-specific involvement of RIG-I in antiviral response.</a> Immunity. 23 (1), 19-28 (2005).</li><li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Anal Biochem. 72, 248-254 (1976).</li><li>Iwamura, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+IRF-3/-7+kinase+and+NF-kappaB+in+response+to+double-stranded+RNA+and+virus+infection:+common+and+unique+pathways.">Induction of IRF-3/-7 kinase and NF-kappaB in response to double-stranded RNA and virus infection: common and unique pathways.</a> Genes Cells. 6 (4), 375-388 (2001).</li><li>tenOever, B. R., Servant, M. J., Grandvaux, N., Lin, R., Hiscott, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognition+of+the+measles+virus+nucleocapsid+as+a+mechanism+of+IRF-3+activation.">Recognition of the measles virus nucleocapsid as a mechanism of IRF-3 activation.</a> J Virol. 76 (8), 3659-3669 (2002).</li><li>Bibeau-Poirier, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+the+I{kappa}B+Kinase+(IKK)-Related+Kinases+Tank-Binding+Kinase+1/IKKi+and+Cullin-Based+Ubiquitin+Ligases+in+IFN+Regulatory+Factor-3+Degradation.">Involvement of the I{kappa}B Kinase (IKK)-Related Kinases Tank-Binding Kinase 1/IKKi and Cullin-Based Ubiquitin Ligases in IFN Regulatory Factor-3 Degradation.</a> J Immunol. 177 (8), 5059-5067 (2006).</li><li>Grandvaux, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustained+Activation+of+Interferon+Regulatory+Factor+3+during+Infection+by+Paramyxoviruses+Requires+MDA5.">Sustained Activation of Interferon Regulatory Factor 3 during Infection by Paramyxoviruses Requires MDA5.</a> J Innate Immun. 6 (5), 650-662 (2014).</li></ol>

The authors declare that they have no competing financial interests.

The protocol we describe here consists of a combination of high-resolution SDS-PAGE and native-PAGE coupled to the use of several phosphospecific antibodies to distinguish the monomeric/dimeric and phosphoforms I-IV of IRF3. Appropriate detection of these IRF3 species is essential to fully characterize IRF3 activation in a specific setting. For instance, LPS stimulation of activated macrophages leads to the formation of dimeric, Ser396/398 phosphorylated IRF3 that exhibits a hypophosphorylated (form II), but not hyperphosphorylated (form III and IV), pattern in SDS-PAGE15. Using the described protocol, this profile can be distinguished from virus-induced hyperphosphorylated forms that require additional Ser339 phosphorylation in addition to Ser386 and Ser396 phosphorylation10. Importantly, this also allows discriminating between Ser396 phosphorylated species that do not exhibit dimerization, but are transcriptionally active10. Importantly, it needs to be noted that the form I-IV pattern of IRF3 phosphoforms applied to human IRF3. Murine IRF3 does not exhibit a similar pattern and therefore activation of IRF3 is characterized only by the use of phosphospecific antibodies in immunoblot after SDS-PAGE and by native-PAGE.
The achievement of a high resolution separation of the distinct phosphorylated species of IRF3 by SDS-PAGE requires specific parameters. For appropriate resolution, it is very important to use gels that have a minimum length of 16 cm. Even with this length of separation gel, it is key to let the migration front reach the bottom of the gel to obtain an appropriate separation of the different IRF3 forms. Also, the stacking gel must extend to a minimum of 1 cm below the comb to obtain well-defined bands. This is important, as the hyperphosphorylated forms of IRF3 migrate very close to one another. Poorly focused bands will result in lack of distinction between phosphorylated forms impairing their respective quantification. Additionally, the concentration of ammonium persulfate and TEMED vary from one SDS-PAGE protocol to another. Variation in these concentrations from the ones indicated here was found to significantly impair the resolution in the separation of the IRF3 species.
Detection of IRF3 dimer formation through the native-PAGE was originally described by the group of Dr. T. Fujita. This protocol uses buffer containing DOC that allows the dissociation of the IRF3 dimer from CBP/p30027. It is highly recommended to proceed to native-PAGE immediately after cell lysis as storage of the WCE at -80 &#176;C was found to result in a significant alteration of the IRF3 monomer/dimer ratio. Additionally, it is very important that the pH of the upper and lower chamber buffers is adjusted to pH 8.4 at RT, and not at 4 &#176;C, after mixing all components to achieve proper separation of the monomer vs dimer. A separation gel with a minimum length of 8.5 cm allows appropriate separation of IRF3 monomer and dimer. The ideal volume of sample plus loading buffer is around 8 &#181;l for a 5 mm well as the resolution is better when the volume is kept to a minimum. Thus, it is important to use the lowest volume of lysis buffer to obtain WCE with a high concentration. However, it should be taken into account that efficient protein extraction requires lysis in at least 5x the volume of the cell pellet. If the volume of the samples exceeds the limit indicated above, this would result in poor resolution of the monomer/dimer. This can be solved by adding a stacking gel containing 4% acrylamide, 62.5 mM Tris-HCl pH 6.8 (RT), 1% ammonium persulfate and 0.1% TEMED in ddH2O. It is very important to load the samples without disturbing banding. Loading slowly with a gel-loading tip close to the bottom of the well gives very good results.
Here, we describe the in vivo detection of phosphorylation at specific residues using currently available phosphospecific antibodies against Ser3869, Ser39619 and Ser39815. The use of IRF3 mutants and mass spectrometry analyses have confirmed that these residues are phosphorylated upon virus infection or overexpression of IKK&#949;/TBK1 kinases and are critical for IRF3 activation6,9,10,19,21. The immunodetection of phospho-Ser396, phospho-Ser398 and total IRF3 after SDS-PAGE requires the use of two identical gels (Figure 1). Indeed, significant loss of sensitivity is observed when phosphospecific antibodies are used after the stripping of the membrane. Therefore, it is highly recommended to use phosphospecific antibodies first. Note that alternative primary and secondary antibodies may also work, but the specific dilutions and sequence of usage should be optimized. For IRF3 analysis, 30 &#181;g of WCE is appropriate to obtain a significant detection of IRF3 phosphorylation by immunoblot using the indicated antibodies when using 4 mm width wells. Although in many cell types infected with various viruses 30 &#181;g was found to be appropriate, this amount might need to be increased depending on cell type and stimulation. It is preferable to use fresh extracts for SDS-PAGE, but the detection of IRF3 phosphorylation using the phosphospecific antibodies described in this paper is stable enough to allow a round of storage of the WCE at -80 &#176;C before analysis. The concentrations of phosphatase inhibitors described here were found to be sufficient when using A549 cells. Higher concentrations were not found to yield better results. However, these concentrations might need to be increased when studying IRF3 activation in cell types that exhibit higher phosphatase activity levels. Importantly, the phosphorylation of IRF3 and the underlying mechanisms are still far from being understood. Therefore, a full panel of Ser/Thr- and Tyr-phosphatase inhibitors is used to block possible yet uncharacterized activities that could affect the detection of IRF3 phosphorylation, dimerization and degradation. For detection of IRF3 monomer and dimer after most virus infections and in most cell types, 8 - 10 &#181;g WCE was found to be sufficient. However, higher amount of proteins might be required for stimulation activating IRF3 less efficiently. The phosphospecific antibodies against Ser386 used in this study are recommended to be used in native-PAGE as the sensitivity in denaturing SDS-PAGE is very weak. Moreover, the phosphorylation at Ser386 is proposed to correlate with the dimeric form of IRF39, thus making the use of these antibodies in native-PAGE an appropriate strategy to confirm this concept in a specific setting. Of note, alternative antibodies are available from various companies and are claimed to efficiently detect phospho-Ser386 after SDS-PAGE. Phosphospecific antibodies against Ser396 have also been efficiently used to detect IRF3 phosphorylation after native-PAGE20. Importantly, Ser402 in the C-terminal cluster of phosphoacceptor sites was also found to be important for IRF3 activation and the phosphorylation of Ser402 was observed through mass spectrometry analysis of IKK&#949;/TBK1-phosphorylated IRF310,21,28. However, despite two different attempts (NG, unpublished), no phosphospecific antibodies are currently available to follow the phosphorylation of this specific residue in vivo. Note that phosphospecific antibodies against Thr157 and Ser339 have also been described11,13. These additional phosphospecific antibodies could be used in a procedure of high-resolution SDS-PAGE similar to the one described here. In this case, additional large SDS-PAGE gels would be required for each antibody and processed the same way as gel #2 (Figure 1). To our knowledge no phosphospecific antibodies against Ser173 or Thr390 have yet been described, but they could be easily added to the protocol described here once they become available14,20.
IRF3 activity is terminated by proteasome-mediated degradation of hyperphosphorylated forms11,17. The protocol described here also permits the monitoring of IRF3 degradation when the kinetic of stimulation is prolonged and is coupled to the use of proteasome inhibitors (MG132, lactacystin or bortezomid). Typically, in A549 cells infected with SeV at 40 HAU/106 cells, IRF3 phosphorylation starts as soon as 2 hr post-SeV infection and IRF3 degradation starts after 12 hr. Proteasome-mediated degradation will be concluded from the observed diminution of forms III and IV levels at late time points that is reversed in conditions with proteasome inhibitors. This will also translate on native-PAGE in a diminution of IRF3 dimer levels that are recovered upon proteasome inhibitor treatment29. Importantly, the high-resolution technique presented here allows differentiating between the degradation process and a lack of activation. Indeed, both degradation and lack of activation of IRF3 result in the absence of detection of dimer/phospho-Ser386/396. However, lack of activation is associated with the detection of IRF3 monomer and of forms I and II, while these IRF3 species are not detected when IRF3 is degraded30.
Immunoprecipitation coupled with mass spectrometry-based analysis is a method of choice to detect the in vivo phosphorylation of IRF3 at distinct sites. This technique was used to confirm the phosphorylation of IRF3 at Ser173, Ser175, Ser386, Thr390, Ser396 and Ser402 residues10,20,21. However, mass spectrometry is not yet an affordable/benchside technique that can be easily used for daily analysis of IRF3 activation following several stimulations at different time points. Therefore, the procedure described here using SDS-PAGE coupled to native-PAGE in combination with immunoblot using total and phosphospecific antibodies constitutes a practical, affordable and sensitive approach to detect all currently defined forms of actived IRF3.

The ubiquitously and constitutively expressed transcription factor Interferon (IFN) Regulatory Factor 3 (IRF3) is critical for the first line of defense against pathogens mainly through the induction of IFN&#946;, but also through the induction of the chemokine (C-C motif) ligand 5 (CCL5) and several antiviral proteins including IFN-induced protein with tetratricopeptide repeats IFIT1/2/31-3. IRF3 activation has been reported following infection with numerous viruses, or exposure to polyinosinic-polycytidylic acid (poly I:C) or lipopolysaccharide (LPS)4. Importantly, most studied viruses have evolved mechanisms to evade the IRF3-mediated response, and thereby escape the host innate immune defense5. Thus, monitoring IRF3 activation is of great importance to understand the molecular mechanisms of the innate antiviral host defense, but also to identify the strategy used by viruses to counteract this response.
Many published reports however provide only a limited analysis of IRF3 activation performed by the monitoring of IRF3-target gene induction (IFNB1 and IFIT1) and/or luciferase reporter gene assay coupled to low resolution sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) analysis of IRF3. However, numerous biochemical studies, analysis of the behavior of various IRF3 mutants and elucidation of IRF3 crystal structure 6-11 have contributed to establish that IRF3 is subjected to a complex set of sequential post-translational modifications by phosphorylation at multiple sites. The set of phosphorylation involved in IRF3 activation appears to be dependent on the stimulus and most likely on the cell type. In uninfected cells, IRF3 coexists as non-phosphorylated and hypophosphorylated species containing phosphoresidues, including Thr135 and Ser173, in the 1-198 aa N-terminal region6,12-14. Accumulation of this hypophosphorylated form of IRF3 is induced by stress inducers, growth factors and DNA-damaging agents6. Phosphorylation of Ser/Thr residues at the C-terminal region of IRF3 containing the transactivation domain is triggered following activation by viruses, poly I:C or LPS in a cell-type dependent manner15-17. C-terminal phosphorylation of IRF3 involves no less than 7 distinct phosphoacceptor sites organized in two main clusters, Ser385/Ser386 and Ser396/Ser398/Ser402/Thr404/Ser405, that each contribute to IRF3 activation through dimerization, nuclear accumulation, association with the CREB-binding protein (CBP)/p300 coactivators, DNA binding to IFN sensitive response element (ISRE) consensus sequences and transactivation of target genes9,10,17-19. Phosphorylation of Thr390 is also thought to contribute to virus-induced IRF3 activation20. Mass spectrometry analyses of IRF3 have demonstrated that Ser386, Thr390, Ser396 and Ser402 residues are directly phosphorylated by the inhibitor of &#954;B kinase &#949; (IKK&#949;)/ TANK-binding kinase 1 (TBK1) kinases9,10. Phosphorylation at the C-terminal residues is also required for termination of IRF3 activation through polyubiquitination and proteasome-mediated degradation10. This process is also dependent on the phosphorylation at Ser339, which is necessary for the recruitment of the propyl isomerase Pin110,11. IRF3 species containing at least phospho-Ser339/386/396 residues are considered hyperphosphorylated forms. The exact sequence and function of each site remains a matter of discussion 10,21. It is now clear that activated IRF3 does not represent a homogeneous state, but that different activated species exhibiting distinct phosphorylation or dimerization characteristics exist 10,22.
To provide a complete understanding of IRF3 activation in response to specific pathogens, it is thus necessary to characterize which of the activated species are induced. Induction of IRF3 target genes, IFNB1 and IFIT1, has proven to provide a reliable read-out for IRF3 activation. However, monitoring expression of these genes does not distinguish between different activation states of IRF3. A comprehensive analysis of IRF3 activation states in a particular setting relies on the detailed characterization of its phosphorylation and dimerization status10. Unphosphorylated (form I), hypophosphorylated (form II) and hyperphosphorylated (forms III and IV) IRF3 forms6,18,23 can be successfully resolved by reduced mobility in high-resolution SDS-PAGE analysis. Monomeric and dimeric IRF3 species can be efficiently identified by native-PAGE analysis. These approaches are greatly improved when used in combination with phosphospecific antibodies directed against distinct IRF3 phosphoacceptor sites.
Standard protocols allow a poor resolution of proteins that does not permit efficient separation of distinct IRF3 phosphorylated forms. Here, we describe in detail a procedure to achieve the highest resolution to monitor the induction of distinct virus-activated IRF3 species using SDS-PAGE coupled to native-PAGE in combination with immunoblot using total and phosphospecific antibodies. In vivo discrimination between the different activated forms of IRF3 is performed based on their mobility shifts observed on SDS-PAGE. Additionally, IRF3 monomer and dimer can be distinguished by non-denaturing electrophoresis. The combination of these two complementary techniques with immunoblot proves to be an affordable and sensitive approach to acquire all the necessary information for a complete analysis of phosphorylation-mediated activation of IRF3.

<table border="0" cellpadding="0" cellspacing="0" width="1098">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">F12/Ham</td>
			<td style="width:191px;">Life Technologies</td>
			<td style="width:119px;">11765-054</td>
			<td style="width:429px;">Warm in a 37&#176;C bath before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Fetal bovine serum</td>
			<td style="width:191px;">Life Technologies</td>
			<td style="width:119px;">12483-020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">L-glutamine</td>
			<td style="width:191px;">Life Technologies</td>
			<td style="width:119px;">25030-081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">D-PBS</td>
			<td style="width:191px;">Life Technologies</td>
			<td style="width:119px;">14190-144</td>
			<td>For cell culture.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Trypsin/EDTA 0.25 %</td>
			<td style="width:191px;">Life Technologies</td>
			<td style="width:119px;">25200-072</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Sendai virus Cantell Strain</td>
			<td style="width:191px;">Charles River Laboratories</td>
			<td style="width:119px;">600503</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Hepes</td>
			<td style="width:191px;">Bioshop</td>
			<td style="width:119px;">HEP001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Sodium chloride (NaCl)</td>
			<td>Bioshop</td>
			<td>SOD001.5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">EDTA</td>
			<td style="width:191px;">Bioshop</td>
			<td style="width:119px;">EDT001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Glycerol</td>
			<td style="width:191px;">Bioshop</td>
			<td style="width:119px;">GLY001.1</td>
			<td style="width:429px;">Cut the extreminity of the tip and pipet slowly as it is very thick.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:359px;">IGEPAL CA-630</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">I7771</td>
			<td style="width:429px;">Registred trademark corresponding to Octylphenoxy poly(ethyleneoxy)ethanol (Nonidet P-40) detergent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Leupeptin</td>
			<td style="width:191px;">Bioshop</td>
			<td style="width:119px;">LEU001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Aprotinin</td>
			<td>Bioshop</td>
			<td>APR600.25&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Sodium fluoride</td>
			<td>Sigma-Aldrich</td>
			<td>201154</td>
			<td></td>
		</tr>
		<tr height="150">
			<td height="150" style="height:150px;">Sodium orthovanadate</td>
			<td>MP Biomedicals</td>
			<td>159664</td>
			<td style="width:429px;">Activation of sodium orthovanadate 0.2M : 1) Ajust the pH to 10.0 using either 1 N NaOH or 1 N HCl. The starting pH of the sodium orthotovanadate solution may vary with lots of chemical. 2) The solution is yellow at pH 10.0. 3) Boil until colorless. 4) Cool to RT. 5) Reajust the pH to 10.0 and repat steps 3-4 until the solution remains colorless and stabilizes at 10.0. Store the activated sodium orthovanadate aliquots at -20&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">p-nitrophenyl phosphate disodium salt hexahydrate</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">P1585</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Beta-Glycerophosphate</td>
			<td>Sigma-Aldrich</td>
			<td>G6376&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Bio-Rad Protein Assay Reagent&#160;</td>
			<td>Bio-Rad</td>
			<td>500-0006&#160;</td>
			<td>Cytotoxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Acrylamide/Bis-Acrylamide (37.5 : 1) 40 %</td>
			<td>Bioshop</td>
			<td>ACR005&#160;</td>
			<td>Cytotoxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Tris-Base</td>
			<td style="width:191px;">Bioshop</td>
			<td>DEO701</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Hydrochloric acid (HCl)</td>
			<td>LabChem</td>
			<td>LC15320-4&#160;</td>
			<td>Work under fume hood. Toxic and irritant.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Sodium dodecyl sulfate (SDS)</td>
			<td>Bioshop</td>
			<td>SDS001.1&#160;</td>
			<td>Irritant.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Amonium persulfate</td>
			<td>Sigma-Aldrich</td>
			<td>A3678</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">TEMED</td>
			<td>Invitrogen</td>
			<td>15524-010</td>
			<td>Toxic and irritant.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Bromophenol blue</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">B392-5</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Beta-mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M6250</td>
			<td style="width:429px;">Work under fume hood. Toxic to the nervous system, mucous membranes. May be toxic to upper respiratory tract, eyes, central nervous system.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Glycine</td>
			<td>Bioshop</td>
			<td>GLN001.5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Sodium deoxycholate</td>
			<td>Sigma-Aldrich</td>
			<td>D6750</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Sodium hydroxide (NaOH)</td>
			<td>Bioshop</td>
			<td>SHY700&#160;</td>
			<td>Irritant.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Nitrocellulose membrane (0.45mm)</td>
			<td style="width:191px;">Bio-Rad</td>
			<td style="width:119px;">162-0115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Acetic acid glacial</td>
			<td>Bioshop</td>
			<td>ACE222.4</td>
			<td>Work under fume hood. Toxic, irritant and flammable.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Red ponceau</td>
			<td>Sigma-Aldrich</td>
			<td>P3504&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Potassium chloride (KCl)</td>
			<td>Sigma-Aldrich</td>
			<td>P3911&#160;</td>
			<td>For PBS composition for immunoblot.</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:359px;">Na2HPO4</td>
			<td>Bioshop</td>
			<td>SPD307.5</td>
			<td>For PBS composition for immunoblot.</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:359px;">KH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>P0662&#160;</td>
			<td>For PBS composition for immunoblot.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Bovine serum albumin</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">A7906</td>
			<td>For PBS-T-BSA composition for immunoblot.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Non-fat dry milk</td>
			<td style="width:191px;">Carnation</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">Poly sorbate 20 (Tween)</td>
			<td>MP Biomedicals</td>
			<td>103168</td>
			<td style="width:429px;">Cut the extreminity of the tip and pipet slowly as it is very thick.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:359px;">Anti-IRF-3-P-Ser386</td>
			<td style="width:191px;">IBL-America</td>
			<td style="width:119px;">18783</td>
			<td>Store aliquoted at -20oC. Avoid freeze/thaw.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:359px;">Anti-IRF-3-P-Ser396</td>
			<td style="width:191px;">Home made19</td>
			<td style="width:119px;"></td>
			<td>Store aliquoted at -80oC. Avoid freeze/thaw.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:359px;">Phospho-IRF-3 (Ser396) (4D4G)</td>
			<td style="width:191px;">Cell Signaling Technology</td>
			<td style="width:119px;">4947s</td>
			<td>Store at -20oC.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:359px;">Anti-IRF-3-P-Ser398</td>
			<td style="width:191px;">Home made15</td>
			<td style="width:119px;"></td>
			<td>Store aliquoted at -80oC. Avoid freeze/thaw.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:359px;">Anti-IRF-3-full length</td>
			<td style="width:191px;">Actif motif</td>
			<td style="width:119px;">39033</td>
			<td>Store aliquoted at -80oC. Avoid freeze/thaw.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:359px;">Anti-IRF3-NES</td>
			<td style="width:191px;">IBL-America</td>
			<td style="width:119px;">18781</td>
			<td>Store aliquoted at -20oC.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Western Lightning Chemiluminescence Reagent Plus</td>
			<td>Perkin-Elmer Life Sciences</td>
			<td>NEL104001EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LAS4000mini CCD camera apparatus</td>
			<td style="width:191px;">GE healthcare</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:359px;">SDS-PAGE Molecular Weight Standards, Broad Range</td>
			<td style="width:191px;">Bio-Rad</td>
			<td style="width:119px;">161-0317</td>
			<td>Store aliquoted at -20oC.</td>
		</tr>
	</tbody>
</table>

a high resolution method to monitor phosphorylation-dependent activation of irf3

The IRF3 transcription factor is critical for the first line of defense against pathogens mainly through interferon &#946; and antiviral gene expression. A detailed analysis of IRF3 activation is essential to understand how pathogens induce or evade the innate antiviral response. Distinct activated forms of IRF3 can be distinguished based on their phosphorylation and monomer vs dimer status. In vivo discrimination between the different activated species of IRF3 can be achieved through the separation of IRF3 phosphorylated forms based on their mobility shifts on SDS-PAGE. Additionally, the levels of IRF3 monomer and dimer can be monitored using non-denaturing electrophoresis. Here, we detail a procedure to reach the highest resolution to gain the most information regarding IRF3 activation status. This is achieved through the combination of a high resolution SDS-PAGE and a native-PAGE coupled to immunoblots using multiple total and phosphospecific antibodies. This experimental strategy constitutes an affordable and sensitive approach to acquire all the necessary information for a complete analysis of the phosphorylation-mediated activation of IRF3.

Figure 2 shows a typical immunoblot image of IRF3 detected with IRF3 total antibodies and IRF3-phosphospecific antibodies against Ser396 and Ser398 after resolution of WCE by high-resolution SDS-PAGE. In unstimulated A549 cells, IRF3 is detected as two bands at 50 and 53 kDa on the SDS-PAGE corresponding to the non-phosphorylated (form I) and the hypophosphorylated (form II) species of IRF3. Exposure of A549 cells to SeV for 3 - 9 hr results in a time-dependent shift to slowly migrating hyperphosphorylated forms III and IV, which are well distinguished from the forms I and II. The hyperphosphorylated bands migrate at 55-57 kDa. The forms III and IV are also specifically immunodetected using the phosphospecific antibodies against Ser396 and Ser398. The immunodetection of actin serves as a control of equal loading between the lanes. A control of SeV infection is shown by the accumulation of the virus nucleocapsid protein (N), which migrates at 60 kDa.
In Figure 3, the profile of detection of IRF3 obtained from WCE resolved by native-PAGE followed by immunoblot using anti-IRF3-NES antibodies and phosphospecific antibodies against Ser386 is shown. In unstimulated A549 cells, IRF3 is detected as a single band corresponding to the monomeric form. Upon infection with SeV for 3 - 9 hr, the levels of IRF3 monomer decrease with a concomitant accumulation of a slowly migrating band that corresponds to the dimeric form of IRF3. The phosphospecific antibodies against Ser386 only detect IRF3 dimer species.
<img alt="Figure 2" src="/files/ftp_upload/53723/53723fig2.jpg" /> 
Figure 2. Detection of Distinct IRF3 Phosphorylated Species (form I-IV) Induced by SeV Infection in A549 Cells. A549 cells were left uninfected or infected with SeV for the indicated times. WCE were analysed by high-resolution SDS-PAGE followed by immunoblot (IB) using anti-IRF3-Ser396 (IRF-3-P-Ser396) and anti-IRF3-Ser398 (IRF-3-P-Ser398) phosphospecific antibodies and anti-IRF3 full length protein antibodies. The infection was monitored using anti-SeV antibodies (the nucleocapsid N protein is shown). Anti-actin antibodies were used as a loading control. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53723/53723fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53723/53723fig3.jpg" /> 
Figure 3. Monitoring of Phosphorylated/dimerized IRF3 Induction by SeV in A549 Cells. A549 cells were left uninfected or infected with SeV for the indicated times. WCE were resolved by native-PAGE and revealed by immunoblot using anti-IRF3-Ser386 (IRF-3-P-Ser386) phosphospecific antibodies and anti-IRF3-NES antibodies. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53723/53723fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A High Resolution Method to Monitor Phosphorylation-dependent Activation of IRF3 at JoVE.com

A High Resolution Method to Monitor Phosphorylation-dependent Activation of IRF3

Here we describe a procedure allowing a detailed analysis of the phosphorylation-dependent activation of the IRF3 transcription factor. This is achieved through the combination of a high resolution SDS-PAGE and a native-PAGE coupled to immunoblots using multiple phosphospecific antibodies.

The IRF3 transcription factor is critical for the first line of defense against pathogens mainly through interferon &#946; and antiviral gene expression. ...

a-high-resolution-method-to-monitor-phosphorylation-dependent

Nanyang Technological University

Research

JoVE Journal

Biology

11.8K Views.  Université de Montréal. Here we describe a procedure allowing a detailed analysis of the phosphorylation-dependent activation of the IRF3 transcription factor. This is achieved through the combination of a high resolution SDS-PAGE and a native-PAGE coupled to immunoblots using multiple phosphospecific antibodies.

Video: A High Resolution Method to Monitor Phosphorylation-dependent Activation of IRF3

High-resolution Measurement of Odor-Driven Behavior in Drosophila Larvae

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral paradigm, the experimenter usually assumes that the rapid diffusion of odorant molecules from the source leads to the creation of a stable gradient in the dish. To establish a quantitative correlation between sensory inputs and behavioral responses, it is necessary to achieve a more thorough characterization of the odorant stimulus conditions. In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral ...

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field. Studying locomotor behavior in Drosophila melanogaster relies on the ability to be able to quantify changes in motion during or in response to a given task. For this reason, a high-resolution video tracking system, such as the one we describe in this paper, is a valuable tool for measuring locomotion in real-time. Our protocol involves the use of an initial air pulse to break the flies momentum, followed by a thirty second filming period in a square chamber. A tracking program is then used to calculate the instantaneous speed of each fly within the chamber in 10 msec increments. Analysis software then compiles this data, and outputs a variety of parameters such as average speed, max speed, time spent in motion, acceleration, etc. This protocol will discuss proper feeding and management of flies for behavioral tasks, handling flies without anesthetization or immobilization, setting up a controlled environment, and running the assay from start to finish.

The study of complex locomotor behavior in Drosophila melanogaster is dependent upon the ability to quantify changes in a given fly's movement. This article demonstrates how to do this using a high-resolution tracking system.

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular biology assays such as synthesis and purification of DNA, diagnostic applications such as quantification of DNA amplification products, and detection of DNA molecules in drug preparations. During this video we will demonstrate the capability of the Hitachi F-7000 Fluorescence Spectrophotometer equipped with a Micro Plate Reader accessory to perform dsDNA quantification using Molecular Probes Quant-it PicoGreen dye reagent kit.
The F-7000 Fluorescence Spectrophotometer offers high sensitivity and high speed measurements. It is a highly flexible system capable of measuring fluorescence, luminescence, and phosphorescence. Several measuring modes are available, including wavelength scan, time scan, photometry and 3-D scan measurement. The spectrophotometer has sensitivity in the range of 50 picomoles of fluorescein when using a 300 &#956;L sample volume in the microplate, and is capable of measuring scan speeds of 60,000 nm/minute. It also has a wide dynamic range of up to 5 orders of magnitude which allows for the use of calibration curves over a wide range of concentrations. The optical system uses all reflective optics for maximum energy and sensitivity. The standard wavelength range is 200 to 750 nm, and can be extended to 900 nm when using one of the optional near infrared photomultipliers. The system allows optional temperature control for the plate reader from 5 to 60 degrees Celsius using an optional external temperature controlled liquid circulator. The microplate reader allows for the use of 96 well microplates, and the measuring speed for 96 wells is less than 60 seconds when using the kinetics mode.
Software controls for the F-7000 and Microplate Reader are also highly flexible. Samples may be set in either column or row formats, and any combination of wells may be chosen for sample measurements. This allows for optimal utilization of the microplate. Additionally, the software allows importing micro plate sample configurations created in Excel and saved in comma separated values, or "csv" format. Microplate measuring configurations can be saved and recalled by the software for convenience and increased productivity. Data results can be output to a standard report, to Excel, or to an optional Report Generator Program.

Demonstration of quantification of dsDNA using Molecular Probes PicoGreen dye and Hitachi F-7000 Fluorescence Spectrophotometer equipped with a microplate reader accessory.

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular ...

Absolute Quantum Yield Measurement of Powder Samples

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and research of illumination, AV equipment, organic EL material, films, filters and fluorescent probes for bio-industry.
Quantum yield is calculated as the ratio of the number of photons absorbed, to the number of photons emitted by a material. The higher the quantum yield, the better the efficiency of the fluorescent material.
For the measurements featured in this video, we will use the Hitachi F-7000 fluorescence spectrophotometer equipped with the Quantum Yield measuring accessory and Report Generator program. All the information provided applies to this system. 
Measurement of quantum yield in powder samples is performed following these steps:
<ol>
 <li>Generation of instrument correction factors for the excitation and emission monochromators. This is an important requirement for the correct measurement of quantum yield. It has been performed in advance for the full measurement range of the instrument and will not be shown in this video due to time limitations.</li>
 <li>Measurement of integrating sphere correction factors. The purpose of this step is to take into consideration reflectivity characteristics of the integrating sphere used for the measurements.</li>
 <li>Reference and Sample measurement using direct excitation and indirect excitation.</li>
 <li>Quantum Yield calculation using Direct and Indirect excitation. Direct excitation is when the sample is facing directly the excitation beam, which would be the normal measurement setup. However, because we use an integrating sphere, a portion of the emitted photons resulting from the sample fluorescence are reflected by the integrating sphere and will re-excite the sample, so we need to take into consideration indirect excitation. This is accomplished by measuring the sample placed in the port facing the emission monochromator, calculating indirect quantum yield and correcting the direct quantum yield calculation.</li>
 <li>Corrected quantum yield calculation.</li>
 <li>Chromaticity coordinates calculation using Report Generator program.</li>
</ol>
The Hitachi F-7000 Quantum Yield Measurement System offer advantages for this
application, as follows:
<ul>
 <li>High sensitivity (S/N ratio 800 or better RMS). Signal is the Raman band of water measured under the following conditions: Ex wavelength 350 nm, band pass Ex and Em 5 nm, response 2 sec), noise is measured at the maximum of the Raman peak. High sensitivity allows measurement of samples even with low quantum yield. Using this system we have measured quantum yields as low as 0.1 for a sample of salicylic acid and as high as 0.8 for a sample of magnesium tungstate.</li>
 <li>Highly accurate measurement with a dynamic range of 6 orders of magnitude allows for measurements of both sharp scattering peaks with high intensity, as well as broad fluorescence peaks of low intensity under the same conditions. </li>
 <li>High measuring throughput and reduced light exposure to the sample, due to a high scanning speed of up to 60,000 nm/minute and automatic shutter function. </li>
 <li>Measurement of quantum yield over a wide wavelength range from 240 to 800 nm.</li>
 <li>Accurate quantum yield measurements are the result of collecting instrument spectral response and integrating sphere correction factors before measuring the sample.</li>
 <li>Large selection of calculated parameters provided by dedicated and easy to use software.
</li>
</ul> 
During this video we will measure sodium salicylate in powder form which is known to have a quantum yield value of 0.4 to 0.5.

In this video we will demonstrate measuring and calculating absolute quantum yield and chromaticity coordinates directly in powder samples using the Hitachi F-7000 Quantum Yield Measuring System.

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and ...

Using High Resolution Computed Tomography to Visualize the Three Dimensional Structure and Function of Plant Vasculature

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being used to evaluate the structure and function of plant xylem network in three dimensions (3D) (e.g. Brodersen et al. 2010; 2011; 2012a,b). HRCT imaging is based on the same principles as medical CT systems, but a high intensity synchrotron x-ray source results in higher spatial resolution and decreased image acquisition time. Here, we demonstrate in detail how synchrotron-based HRCT (performed at the Advanced Light Source-LBNL Berkeley, CA, USA) in combination with Avizo software (VSG Inc., Burlington, MA, USA) is being used to explore plant xylem in excised tissue and living plants. This new imaging tool allows users to move beyond traditional static, 2D light or electron micrographs and study samples using virtual serial sections in any plane. An infinite number of slices in any orientation can be made on the same sample, a feature that is physically impossible using traditional microscopy methods.
Results demonstrate that HRCT can be applied to both herbaceous and woody plant species, and a range of plant organs (i.e. leaves, petioles, stems, trunks, roots). Figures presented here help demonstrate both a range of representative plant vascular anatomy and the type of detail extracted from HRCT datasets, including scans for coast redwood (Sequoia sempervirens), walnut (Juglans spp.), oak (Quercus spp.), and maple (Acer spp.) tree saplings to sunflowers (Helianthus annuus), grapevines (Vitis spp.), and ferns (Pteridium aquilinum and Woodwardia fimbriata). Excised and dried samples from woody species are easiest to scan and typically yield the best images. However, recent improvements (i.e. more rapid scans and sample stabilization) have made it possible to use this visualization technique on green tissues (e.g. petioles) and in living plants. On occasion some shrinkage of hydrated green plant tissues will cause images to blur and methods to avoid these issues are described. These recent advances with HRCT provide promising new insights into plant vascular function.

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique that can be used to study the structure and function of plant vasculature in 3D. We demonstrate how HRCT facilitates exploration of xylem networks across a wide range of plant tissues and species.

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being ...

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

An ex vivo technique for imaging mouse airway epithelia for quantitative analysis of motile cilia function important for insight into mucociliary clearance function has been established. Freshly harvested mouse trachea is cut longitudinally through the trachealis muscle and mounted in a shallow walled chamber on a glass-bottomed dish. The trachea sample is positioned along its long axis to take advantage of the trachealis muscle to curl longitudinally. This allows imaging of ciliary motion in the profile view along the entire tracheal length. Videos at 200 frames/sec are obtained using differential interference contrast microscopy and a high speed digital camera to allow quantitative analysis of cilia beat frequency and ciliary waveform. With the addition of fluorescent beads during imaging, cilia generated fluid flow also can be determined. The protocol time spans approximately 30 min, with 5 min for chamber preparation, 5-10 min for sample mounting, and 10-15 min for videomicroscopy.

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

An easy and reliable technique for visualizing and quantifying airway cilia motility and cilia generated flow using mouse trachea is described. This technique can be modified to determine how a wide range of factors influence cilia motility, including pharmacological agents, genetic factors, environmental exposures, and/or mechanical factors such as mucus load.

An ex vivo technique for imaging mouse airway epithelia for quantitative  analysis of motile cilia function important for insight into mucociliary  ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

High-resolution Optical Mapping of the Mouse Sino-atrial Node

Sino-atrial node (SAN) dysfunctions and associated complications constitute important causes of morbidity in patients with cardiac diseases. The development of novel pharmacological therapies to cure these patients relies on the thorough understanding of both normal physiology and pathophysiology of the SAN. Among the studies of cardiac pacemaking, the mouse SAN is widely used due to its feasibility for modifications in the expression of different genes that encode SAN ion channels or calcium handling proteins. Emerging evidence from electrophysiological and histological studies has also proved the representativeness and similarity of the mouse SAN structure and functions to larger mammals, including the presence of specialized conduction pathways from the SAN to the atrium and a complex pacemakers' hierarchy within the SAN. Recently, the technique of optical mapping has greatly facilitated the exploration and investigation of the origin of excitation and conduction within and from the mouse SAN, which in turn has extended the understanding of the SAN and benefited clinical treatments of SAN dysfunction associated diseases. In this manuscript, we have described in detail how to perform the optical mapping of the mouse SAN from the intact, Langendorff-perfused heart and from the isolated atrial preparation. This protocol is a useful tool to enhance the understanding of mouse SAN physiology and pathophysiology.

Here, we present a protocol for optical mapping of electrical activity from the mouse right atrium and especially the sino-atrial node, at a high spatial and temporal resolution.