The work was supported by funding from the Croucher Foundation and City University startup funds. We thank all members of the Chow laboratory for help with the experiment and critical reading of the manuscript.

NOTE: The protocol described here uses CAL-1 pDC cell line treated with resiquimod (R848), an agonist for TLR7/8. This protocol has been applied to other human and murine cell types, including RAW 264.7 (murine macrophage line), THP-1 (human monocytic cell line), BJAB (human B cell line), Ramos (human B cell line), and MUTZ-3 (human dendritic cell line)23.
1. Stimulation of CAL-1 Cells
<ol>
	<li>Maintain CAL-1 cell cultures in a T75 flask at 37 &#176;C and 5% CO2 under sterile conditions with 20&#8722;25 mL of RPMI 1640 medium containing 5% fetal bovine serum (FBS), 25 mM HEPES, and 1x mercaptoethanol (i.e., complete RPMI 1640 medium).</li>
	<li>Transfer the cells into a 50 mL conical tube. 
	NOTE: CAL-1 cells are non-adherent. For adherent cell types, standard trypsinization can be performed to harvest cells.</li>
	<li>Centrifuge the cells at 200 x g for 5 min at room temperature (RT). Remove supernatant and resuspend the cell pellet in 8 mL of the complete RPMI 1640 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 well in a 6 well plate with 4 mL of preheated complete RPMI 1640 medium in each well. Incubate for 20&#8722;24 h at 37 &#176;C and 5% CO2 to allow the confluency to reach 90%&#8722;95% (corresponding to approximately 1.5 x 106 cells).</li>
	<li>Stimulate the cells by adding 4 &#181;L of 1 mg/mL R848 per well of the 6 well plate (final concentration of 1 &#181;g/mL). Also set up an unstimulated control well with cells without the R848 treatment.</li>
	<li>Ensure the R848 is evenly dispersed by gently rocking the plate side to side. Then, incubate the cells for 2&#8722;16 h in the incubator at 37 &#176;C and 5% CO2.</li>
</ol>
2. Extraction of Cellular Proteins
<ol>
	<li>Transfer the cell suspensions from the 6 well plate into 5 mL centrifuge tubes.</li>
	<li>Centrifuge at 200 x g for 5 min at RT. Remove the supernatant and resuspend the cell pellet in 1 mL of phosphate-buffered saline (PBS) to obtain a homogeneous single cell suspension.</li>
	<li>Transfer the cell suspension into a 1.5 mL centrifuge tube.</li>
	<li>Spin down briefly at 12,000 x g for 0.5&#8722;1 min at 4 &#176;C and carefully remove the supernatant.</li>
	<li>Prepare the lysis buffer containing 6.25 mL of 1 M Tris-HCl pH 7.4 (final concentration of 25 mM), 7.5 mL of 5 M NaCl (final concentration of 150 mM), 0.5 mL of 0.5 M EDTA (final concentration of 1 mM), 2.5 mL of NP-40 (final concentration of 1%) and 7.5 mL of glycerol (final concentration of 5%) in 250 mL of deionized water (ddH2O). Add 100x protease inhibitor single-use cocktail to a final concentration of 1x to the lysis buffer just before use. Keep the prepared lysis buffer on ice. 
	NOTE: The lysis buffer without the protease can be stored at 4 &#176;C.</li>
	<li>Resuspend the cell pellet in 30 &#181;L of ice-cold lysis buffer and mix by pipetting up and down.</li>
	<li>Incubate on ice for 15&#8722;20 min.</li>
	<li>Clarify the lysate by centrifuging at 12,000 x g for 15&#8722;20 min at 4 &#176;C. Transfer the supernatant into a new prechilled 1.5 mL centrifuge tube. Keep the extracts on ice at all times. 
	NOTE: Cell lysates can be stored at -20 &#176;C or -80 &#176;C. Do not boil the samples.</li>
	<li>Measure protein concentration using Bradford reagent.</li>
</ol>
3. Analysis of IRF5 Dimerization by Native PAGE
<ol>
	<li>Prepare the upper (-) and lower (+) chamber electrophoresis buffers. The upper chamber buffer consists of 0.3% sodium deoxycholate (NaDOC) in 1x native PAGE running buffer, and the lower chamber consists of only 1x native PAGE running buffer. 
	NOTE: Prepare a fresh upper chamber buffer for every new run.</li>
	<li>Rinse a 3%&#8722;12% native PAGE gel thoroughly with water without distorting the wells. Set the gel into the mini gel tank and remove the comb. Prerun the gel in a 4 &#176;C cold room or on ice at 150 V for 30 min. 
	NOTE: Prerunning removes excessive ammonia and persulfate ions that can interfere with the running of the gel.</li>
	<li>During the prerun, prepare the samples for loading by mixing the cellular proteins kept on ice with 4x native sample buffer.</li>
	<li>After the prerun, load 10&#8722;15 &#181;g of protein with a final volume of 10&#8722;15 &#181;L per sample. 
	NOTE: Overloading of protein can cause smearing.</li>
	<li>Run gel at 85 V for 30 min, then 150 V for 2 h. 
	NOTE: Considering differences in equipment and cell lines used by different labs, minor modifications to the concentration of protein samples, voltage and running time may be appropriate to optimize this protocol. Lowering pre-running and running voltage while increasing running time may help to improve dimer resolution and result consistency.</li>
	<li>Soak the gel in SDS running buffer (25 mM Tris pH 8.3, 250 mM Glycine, 0.1% SDS) for 30 min at RT. 
	NOTE: No agitation is required. Occasionally, the intensity of the band may not be proportional to the amount of protein loaded due to inefficient transfer in the presence of deoxycholate (DOC), which mainly affects the monomeric form of IRF5. Soaking the gel in SDS running buffer prior to the transfer solves this problem. The gel is fragile. Handle with extreme care from the bottom (i.e., higher percentage) end of the gel.</li>
</ol>
4. Immunoblot Analysis of IRF5
<ol>
	<li>Activate the polyvinylidene difluoride (PDVF) membrane by soaking it in methanol for approximately 5 min.</li>
	<li>Make a cut on one corner of the membrane to indicate its orientation. Assemble the transfer sandwich according to the sequential order detailed in the manufacturer&#8217;s protocol with extra care to ensure that no air bubbles are trapped within.</li>
	<li>Place the transfer cassette into the tank and transfer at 20 V for 1 h on ice. 
	NOTE: Perform all incubations and washes in subsequent steps with a rocking shaker.</li>
	<li>Remove the membrane from the cassette with plastic forceps after the transfer is completed. Block the membrane in blocking buffer (TBS) for 45 min at RT. 
	NOTE: TBS buffer with 5% BSA can also be used as a blocking buffer.</li>
	<li>Incubate the membrane with the primary antibody listed in Table 1. Wash the membrane with 1x TBST washing buffer (20 mM Tris, pH 7.0, 150 mM NaCl and 0.1% Tween 20) for 3 min while rocking. Repeat the wash 2x.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Dillution</td>
			<td>Dillution buffer</td>
			<td>Incubation</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Primary antibody(Anti-IRF5)</td>
			<td>1/1,000</td>
			<td>TBS blocking buffer</td>
			<td>Overnight at 4 &#176;C or 2 h at RT</td>
			<td>Diluted antibodies can be reused several times if stored at 4 &#176;C in the presence of 0.02% sodium azide.</td>
		</tr>
		<tr>
			<td>Secondary antibody(Anti-rabbit)</td>
			<td>1/10,000</td>
			<td>TBS blocking buffer</td>
			<td>45 min at RT</td>
			<td>Diluted antibodies can be reused several times if stored at 4 &#176;C in the presence of 0.02% sodium azide.</td>
		</tr>
		<tr>
			<td colspan="5">NOTE: Dilution need to be optimized as it varies between manufacturers.</td>
		</tr>
	</tbody>
</table>
Table 1: Specifications of the antibodies used in the immunoblotting procedure.
<ol start="6">
	<li>Incubate the membrane with the secondary antibody listed in Table 1. Wash the membranes for 3 min in 1x TBST washing buffer. Repeat the wash 2x.</li>
	<li>Scan the blot using an appropriate gel documentation system.</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IKKalpha+negatively+regulates+IRF-5+function+in+a+MyD88-TRAF6+pathway.">IKKalpha negatively regulates IRF-5 function in a MyD88-TRAF6 pathway.</a> Cellular Signalling. 22 (1), 117-127 (2010).</li><li>Paun, 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=Functional+characterization+of+murine+interferon+regulatory+factor+5+(IRF-5)+and+its+role+in+the+innate+antiviral+response.">Functional characterization of murine interferon regulatory factor 5 (IRF-5) and its role in the innate antiviral response.</a> Journal of Biological Chemistry. 283 (21), 14295-14308 (2008).</li><li>Yasuda, 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=Murine+dendritic+cell+type+I+IFN+production+induced+by+human+IgG-RNA+immune+complexes+is+IFN+regulatory+factor+(IRF)5+and+IRF7+dependent+and+is+required+for+IL-6+production.">Murine dendritic cell type I IFN production induced by human IgG-RNA immune complexes is IFN regulatory factor (IRF)5 and IRF7 dependent and is required for IL-6 production.</a> The Journal of Immunology. 178 (11), 6876-6885 (2007).</li><li>Steinhagen, F., 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=IRF-5+and+NF-kappaB+p50+co-regulate+IFN-beta+and+IL-6+expression+in+TLR9-stimulated+human+plasmacytoid+dendritic+cells.">IRF-5 and NF-kappaB p50 co-regulate IFN-beta and IL-6 expression in TLR9-stimulated human plasmacytoid dendritic cells.</a> European Journal of Immunology. 43 (7), 1896-1906 (2013).</li><li>Gratz, 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=Type+I+interferon+production+induced+by+Streptococcus+pyogenes-derived+nucleic+acids+is+required+for+host+protection.">Type I interferon production induced by Streptococcus pyogenes-derived nucleic acids is required for host protection.</a> PLoS Pathogens. 7 (5), 1001345 (2011).</li></ol>

The authors have nothing to disclose.

The protocol described here is a modified native PAGE that distinguishes both monomeric and dimeric forms of endogenous IRF5. There have been few studies reporting the detection of endogenous IRF5 activation using the specialized imaging flow cytometry technique23,27,28,30. This protocol uses a common technique and commonplace reagents and tools to assess the endogenous IRF5 activation state du...

Interferon regulatory factor 5 (IRF5) is an important transcription regulator that plays a prominent role in regulating the immune response, particularly in the release of pro-inflammatory cytokines and type I interferons (IFNs)1,2,3. Misregulation of IRF5 is a contributing factor in numerous autoimmune diseases, as evident by various polymorphisms in the IRF5 locus that are associated with systemic lupus erythematosus, multiple sclerosis, rheumatoid arthritis, etc.4,5,6,7,8,9,10. Therefore, a robust detection assay for endogenous IRF5 activation state is crucial for understanding the regulatory pathways and downstream effects of IRF5 in a physiologically relevant cellular context.
IRF5 is constitutively expressed in monocytes, dendritic cells (DCs), B cells, and macrophages1,11. As with other IRF family transcription factors, IRF5 resides in the cytoplasm in its latent state. Upon activation, IRF5 is phosphorylated and forms homodimers, which then translocate into the nucleus and bind to specific regulatory elements of genes encoding type I IFNs and pro-inflammatory cytokines, eventually inducing the expression of these genes1,2,11,12,13. IRF5 regulates the innate immune responses downstream of various Toll-like receptors (TLRs), such as TLR7, TLR 8, and TLR 9, which are localized in endosomes and use MyD88 for signaling1,11,14. These TLRs primarily recognize foreign nucleic acid species such as single-stranded RNA (ssRNA) and unmethylated CpG DNA that are symptomatic of an infection15,16,17,18. IRF5 has been shown to regulate immune responses against bacterial, viral, and fungal infections19,20,21. Considering IRF5&rsquo;s influential and diverse role in the immune system, enhancing or dampening IRF5 activity could serve as a novel avenue for the development of therapeutic agents22. Hence, it is critical to develop a protocol to monitor the activation status of endogenous IRF5 to allow thorough investigation of the pathways and mechanisms regulating IRF5 activity in different cell types.
To the best of our knowledge, no biochemical or gel electrophoretic assay for endogenous IRF5 activation has been published prior to the development of this protocol. Phosphorylation has been shown to be an important first step of IRF5 activation, and a phosphospecific IRF5 antibody was developed that led to the discovery and confirmation of a serine residue important for IRF5 activity13. However, while the antibody clearly detects phosphorylated IRF5 when immunoprecipitated or overexpressed23, it fails to detect IRF5 phosphorylation in a whole cell lysate in our hands (data not shown). Dimerization is the next step of IRF5 activation, and many important studies to date investigating this step relied on overexpression of epitope-tagged IRF5, often in irrelevant cell types that do not normally express IRF511,12,24,25. Previous studies have shown that dimerized IRF5 may not always translocate into the nucleus and hence is not necessarily fully activated25,26. An assay for endogenous IRF5 nuclear localization was developed to assess IRF5 activation by imaging flow cytometry27. This assay has been applied in studies that were crucial to understanding IRF5 activity, especially in primary or rare cell types28,29 and greatly advanced the knowledge in the field. However, this assay relies on a specialized instrument that is not widely available to researchers. Further, it is often necessary to investigate the initial steps of activation while dissecting IRF5 regulatory pathways and identifying upstream regulators and pathway components. This study provides a robust and reliable biochemical assay for the early activation events of IRF5 that can be performed in labs equipped with molecular biology tools. The protocol described here will be very useful in investigating the pathways and mechanisms of IRF5 actions, especially when combined with orthogonal assays such as the imaging flow cytometric analysis of IRF5 nuclear localization23,27,28,30.
Native polyacrylamide gel electrophoresis (native PAGE) is a widely used method to analyze protein complexes31,32. Unlike sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), native PAGE separates proteins on the basis of their shape, size, and charge. It also retains native protein structure without denaturation31,33,34,35. The protocol presented takes advantage of these features of native PAGE and detects both monomeric and dimeric forms of IRF5. This method is particularly important for detecting early activation events because there is no suitable commercially available antibody that can detect endogenous phosphorylated IRF5. Previously, several published studies used native PAGE to assess IRF5 dimerization. However, the majority of these studies depended on the overexpression of exogenous epitope-tagged IRF5 to analyze activation status2,13,24,36,37. This work presents a step-by-step protocol for analyzing endogenous IRF5 dimerization via a modified native PAGE technique in a human plasmacytoid dendritic cell (pDC) line, where IRF5 activity has been shown to be crucial for its function1,38,39,40. This same technique has been applied to other cell lines23.

<table><tbody><tr><td>2-Mercaptoethanol</td><td>Life Technologies, HK</td><td>21985023</td><td></td></tr><tr><td>300 W/250 V power supply 230 V AC</td><td>Life Technologies, HK</td><td>PS0301</td><td></td></tr><tr><td>Anti-IRF5 antibody</td><td>Bethyl Laboratories, USA</td><td>A303-385</td><td></td></tr><tr><td>BIOSAN Rocker Shaker (cold room safe)</td><td>EcoLife, HK</td><td>MR-12</td><td></td></tr><tr><td>EDTA Buffer, pH 8, 0.5 M 4x 100 mL</td><td>Life Technologies</td><td>15575020</td><td></td></tr><tr><td>Glycerol 500 mL</td><td>Life Technologies</td><td>15514011</td><td></td></tr><tr><td>Glycine</td><td>Life Technologies, HK</td><td>15527013</td><td></td></tr><tr><td>Goat anti-Mouse IgG DyLight 800 Conjugated Antibody</td><td>LAB-A-PORTER/Rockland, HK</td><td>610-145-002-0.5</td><td></td></tr><tr><td>Goat anti-Rabbit IgG DyLight 800 Conjugated Antibody</td><td>LAB-A-PORTER/Rockland, HK</td><td>611-145-002-0.5</td><td></td></tr><tr><td>Halt protease inhibitor cocktail (100x)</td><td>Thermo Fisher Scientific, HK</td><td>78430</td><td></td></tr><tr><td>HEPES</td><td>Life Technologies, HK</td><td>15630080</td><td></td></tr><tr><td>LI-COR Odyssey Blocking Buffer (TBS)</td><td>Gene Company, HK</td><td>927-50000</td><td></td></tr><tr><td>Mini Tank blot module combo; Transfer module, accessories</td><td>Life Technologies, HK</td><td>NW2000</td><td></td></tr><tr><td>NativePAGE 3-12% gels, 10 well kit</td><td>Life Technologies, HK</td><td>BN1001BOX</td><td></td></tr><tr><td>NativePAGE Running Buffer 20x</td><td>Life Technologies, HK</td><td>BN2001</td><td></td></tr><tr><td>NativePAGE Sample Buffer 4x</td><td>Life Technologies, HK</td><td>BN2003</td><td></td></tr><tr><td>NP-40 Alternative, Nonylphenyl Polyethylene Glycol</td><td>Tin Hang/Calbiochem, HK</td><td>#492016-100ML</td><td></td></tr><tr><td>PBS 7.4</td><td>Life Technologies, HK</td><td>10010023</td><td></td></tr><tr><td>Polyvinylidene difluoride (PVDF) membrane</td><td>Bio-gene/Merck Millipore, HK</td><td>IPFL00010</td><td></td></tr><tr><td>Protein assay kit II (BSA)</td><td>Bio-Rad, HK</td><td>5000002</td><td></td></tr><tr><td>R848</td><td>Invivogen, HK</td><td>tlrl-r848</td><td></td></tr><tr><td>RPMI 1640</td><td>Life Technologies, HK</td><td>61870127</td><td></td></tr><tr><td>Sodium Chloride</td><td>ThermoFisher</td><td>BP358-1</td><td></td></tr><tr><td>Sodium deoxycholate &amp;ge;97% (titration)</td><td>Tin Hang/Sigma, HK</td><td>D6750-100G</td><td></td></tr><tr><td>Tris</td><td>Life Technologies, HK</td><td>15504020</td><td></td></tr><tr><td>TWEEN 20</td><td>Tin Hang/Sigma, HK</td><td>#P9416-100ML</td><td></td></tr></tbody></table>

native polyacrylamide gel electrophoresis immunoblot analysis of endogenous irf5 dimerization

Interferon regulatory factor 5 (IRF5) is a key transcription factor for regulating the immune response. It is activated downstream of the Toll-like receptor myeloid differentiation primary response gene 88 (TLR-MyD88) signaling pathway. IRF5 activation involves phosphorylation, dimerization, and subsequent translocation from the cytoplasm into the nucleus, which in turn induces the gene expression of various pro-inflammatory cytokines. A detection assay for IRF5 activation is essential to studying IRF5 functions and its relevant pathways. This article describes a robust assay to detect endogenous IRF5 activation in the CAL-1 human plasmacytoid dendritic cell (pDC) line. The protocol consists of a modified nondenaturing electrophoresis assay that can distinguish IRF5 in its monomer and dimer forms, thus providing an affordable and sensitive approach to analyze IRF5 activation.

The immunoblot (IB) with an anti-IRF5 antibody was performed on CAL-1 cells unstimulated or stimulated with 1 &#181;g/mL R848 for 2 h (Figure 1). Cell lysates were prepared, and the native PAGE was performed. In unstimulated CAL-1 cells, IRF5 was detected as a single band on the native PAGE, corresponding to its monomeric form. Upon treatment of CAL-1 cells with R848 for 2 h, the level of IRF5 monomer decreased with a concurrent increase in the accumulation of a slowly migrating band that co...

Watch this Scientific Journal Video about Native Polyacrylamide Gel Electrophoresis Immunoblot Analysis of Endogenous IRF5 Dimerization at JoVE.com

Native Polyacrylamide Gel Electrophoresis Immunoblot Analysis of Endogenous IRF5 Dimerization

A native Western blot method for analyzing endogenous interferon regulatory factor 5 dimerization in the CAL-1 plasmacytoid dendritic cell line is described. This protocol can be applied to other cell lines as well.

Interferon regulatory factor 5 (IRF5) is a key transcription factor for regulating the immune response. It is activated downstream of the Toll-like ...

native-polyacrylamide-gel-electrophoresis-immunoblot-analysis

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Cell Biology

Immunology and Infection

Unit 1

Analyzing Cells and Proteins

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9.8K Views.  City University of Hong Kong. A native Western blot method for analyzing endogenous interferon regulatory factor 5 dimerization in the CAL-1 plasmacytoid dendritic cell line is described. This protocol can be applied to other cell lines as well.

Video: Native Polyacrylamide Gel Electrophoresis Immunoblot Analysis of Endogenous IRF5 Dimerization

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor (IRIS)

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, environmental/agricultural/biodefense monitoring, nanobiotechnology, and more. For diagnostic applications, monitoring (detecting) the presence, absence, or abnormal expression of targeted proteomic or genomic biomarkers found in patient samples can be used to determine treatment approaches or therapy efficacy. In the research arena, information on molecular affinities and specificities are useful for fully characterizing the systems under investigation.
Many of the current systems employed to determine molecular concentrations or affinities rely on the use of labels. Examples of these systems include immunoassays such as the enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) techniques, gel electrophoresis assays, and mass spectrometry (MS). Generally, these labels are fluorescent, radiological, or colorimetric in nature and are directly or indirectly attached to the molecular target of interest. Though the use of labels is widely accepted and has some benefits, there are drawbacks which are stimulating the development of new label-free methods for measuring these interactions. These drawbacks include practical facets such as increased assay cost, reagent lifespan and usability, storage and safety concerns, wasted time and effort in labelling, and variability among the different reagents due to the labelling processes or labels themselves. On a scientific research basis, the use of these labels can also introduce difficulties such as concerns with effects on protein functionality/structure due to the presence of the attached labels and the inability to directly measure the interactions in real time.
Presented here is the use of a new label-free optical biosensor that is amenable to microarray studies, termed the Interferometric Reflectance Imaging Sensor (IRIS), for detecting proteins, DNA, antigenic material, whole pathogens (virions) and other biological material. The IRIS system has been demonstrated to have high sensitivity, precision, and reproducibility for different biomolecular interactions [1-3]. Benefits include multiplex imaging capacity, real time and endpoint measurement capabilities, and other high-throughput attributes such as reduced reagent consumption and a reduction in assay times. Additionally, the IRIS platform is simple to use, requires inexpensive equipment, and utilizes silicon-based solid phase assay components making it compatible with many contemporary surface chemistry approaches.
Here, we present the use of the IRIS system from preparation of probe arrays to incubation and measurement of target binding to analysis of the results in an endpoint format. The model system will be the capture of target antibodies which are specific for human serum albumin (HSA) on HSA-spotted substrates.

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor IRIS

Quantitative, high-throughput, real-time, and label-free biomolecular detection (DNA, protein, etc.) on SiO2 surfaces can be achieved using a simple interferometric technique which relies on LED illumination, minimal optical components, and a camera. The Interferometric Reflectance Imaging Sensor (IRIS) is inexpensive, simple to use, and amenable to microarray formats.

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, ...

Bioengineering

An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. Radioactivity has been the predominant method of DNA labeling in EMSAs. However, recent advances in fluorescent dyes and scanning methods have prompted the use of fluorescent tagging of DNA as an alternative to radioactivity for the advantages of easy handling, saving time, reducing cost, and improving safety. We have recently used fluorescent EMSA (fEMSA) to successfully address an important biological question. Our fEMSA analysis provides mechanistic insight into the effect of a missense mutation, G73E, in the highly conserved HMG transcription factor SOX-2 on olfactory neuron type diversification. We found that mutant SOX-2G73E protein alters specific DNA binding activity, thereby causing olfactory neuron identity transformation. Here, we present an optimized and cost-effective step-by-step protocol for fEMSA using infrared fluorescent dye-labeled oligonucleotides containing the LIM-4/SOX-2 adjacent target sites and purified SOX-2 proteins (WT and mutant SOX-2G73E proteins) as a biological example.

We describe here an optimized protocol of fluorescent Electrophoretic Mobility Shift Assays (fEMSA) using purified SOX-2 proteins together with infrared fluorescent dye-labeled DNA probes as a case study to tackle an important biological question.

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. ...

Biochemistry

Monitoring Activation of the Antiviral Pattern Recognition Receptors RIG-I And PKR By Limited Protease Digestion and Native PAGE

Host defenses to virus infection are dependent on a rapid detection by pattern recognition receptors (PRRs) of the innate immune system. In the cytoplasm, the PRRs RIG-I and PKR bind to specific viral RNA ligands. This first mediates conformational switching and oligomerization, and then enables activation of an antiviral interferon response. While methods to measure antiviral host gene expression are well established, methods to directly monitor the activation states of RIG-I and PKR are only partially and less well established.
Here, we describe two methods to monitor RIG-I and PKR stimulation upon infection with an established interferon inducer, the Rift Valley fever virus mutant clone 13 (Cl 13). Limited trypsin digestion allows to analyze alterations in protease sensitivity, indicating conformational changes of the PRRs. Trypsin digestion of lysates from mock infected cells results in a rapid degradation of RIG-I and PKR, whereas Cl 13 infection leads to the emergence of a protease-resistant RIG-I fragment. Also PKR shows a virus-induced partial resistance to trypsin digestion, which coincides with its hallmark phosphorylation at Thr 446. The formation of RIG-I and PKR oligomers was validated by native polyacrylamide gel electrophoresis (PAGE). Upon infection, there is a strong accumulation of RIG-I and PKR oligomeric complexes, whereas these proteins remained as monomers in mock infected samples.
Limited protease digestion and native PAGE, both coupled to western blot analysis, allow a sensitive and direct measurement of two diverse steps of RIG-I and PKR activation. These techniques are relatively easy and quick to perform and do not require expensive equipment.

Innate defenses to virus infections are triggered by pattern recognition receptors (PRRs). The two cytoplasmic PRRs RIG-I and PKR bind to viral signature RNAs, change conformation, oligomerize, and activate antiviral signaling. Methods are described which allow to conveniently monitor the conformational switching and the oligomerization of these cytoplasmic PRRs.

Host defenses to virus infection are dependent on a rapid detection by pattern recognition receptors (PRRs) of the innate immune system. In the cytoplasm, ...

Vasodilation of Isolated Vessels and the Isolation of the Extracellular Matrix of Tight-skin Mice

The interferon regulatory factor 5 (IRF5) is crucial for cells to determine if they respond in a pro-inflammatory or anti-inflammatory fashion. IRF5's ability to switch cells from one pathway to another is highly attractive as a therapeutic target. We designed a decoy peptide IRF5D with a molecular modeling software for designing small molecules and peptides.
IRF5D inhibited IRF5, reduced alterations in extracellular matrix, and improved endothelial vasodilation in the tight-skin mouse (Tsk/+). The Kd of IRF5D for recombinant IRF5 is 3.72 &plusmn; 0.74 x 10-6 M as determined by binding experiments using biolayer interferometry experiments. Endothelial cells (EC) proliferation and apoptosis were unchanged using increasing concentrations of IRF5D (0 to 100 &#181;g/mL, 24 h). Tsk/+ mice were treated with IRF5D (1 mg/kg/d subcutaneously, 21 d). IRF5 and ICAM expressions were decreased after IRF5D treatment. Endothelial function was improved as assessed by vasodilation of facialis arteries from Tsk/+ mice treated with IRF5D compared to Tsk/+ mice without IRF5D treatment. As a transcription factor, IRF5 traffics from the cytosol to the nucleus. Translocation was assessed by immunohistochemistry on cardiac myocytes cultured on the different cardiac extracellular matrices. IRF5D treatment of the Tsk/+ mouse resulted in a reduced number of IRF5 positive nuclei in comparison to the animals without IRF5D treatment (50 &#181;g/mL, 24 h). These findings demonstrate the important role that IRF5 plays in inflammation and fibrosis in Tsk/+ mice.

We describe the isolation of cardiac extracellular matrix from C57Bl/6J control mice, tight-skin mice, and tight-skin mice treated with the IRF5 inhibitory peptide. We also describe the vasodilation studies on the isolated vessels from C57Bl/6J, tight-skin mice and tight-skin mice treated with the IRF5 inhibitory peptide.

The interferon regulatory factor 5 (IRF5) is crucial for cells to determine if they respond in a pro-inflammatory or anti-inflammatory fashion. IRF5's ...

Predicting Amino Acid Preferences of Protein-Protein Binding Interfaces

Computational Prediction of Amino Acid Preferences of Potentially Multispecific Peptide-Binding Domains Involved in Protein-Protein Interactions

Many protein-protein interactions involve the binding of short protein segments to peptide-binding domains. Usually, such interactions require the recognition of linear motifs with variable conservation. The combination of highly conserved and more variable regions in the same ligands often contributes to the multispecificity of binding, a common property of enzymes and cell signaling proteins. Characterization of amino acid preferences of peptide-binding domains is important for the design of mediators of protein-protein interactions (PPIs). Computational methods are an efficient alternative to the often costly and cumbersome experimental techniques, enabling the design of potential mediators that can be later validated in downstream experiments. Here, we described a methodology using the Pepspec application of the Rosetta molecular modeling package to predict the amino acid preferences of peptide-binding domains. This methodology is useful when the structure of the receptor protein and the nature of the peptide ligand are both known or can be inferred. The methodology starts with a well-characterized anchor from the ligand, which is extended by randomly adding amino acid residues. The binding affinity of peptides generated this way is then evaluated by flexible-backbone peptide docking in order to select the peptides with the best predicted binding scores. These peptides are then used to calculate amino acid preferences and to optionally compute a position-weight matrix (PWM) that can be used in further studies. To illustrate the application of this methodology, we used the interaction between subunits of human interferon regulatory factor 5 (IRF5), previously known to be multispecific but globally guided by a short conserved motif called pLxIS. The estimated amino acid preferences were consistent with previous knowledge about the IRF5 binding surface. Positions occupied by phosphorylatable serine residues exhibited a high frequency of aspartate and glutamate, likely because their negatively charged side chains are similar to phosphoserine.

Author Spotlight: A Computational Approach to Decipher Amino Acid Preferences in Multispecific Protein-Protein Interactions

We describe a methodology based on sequence diversification to estimate the amino acid preferences of multispecific binding sites in protein-protein interactions (PPIs). In this strategy, thousands of potential peptide ligands are generated and screened in silico, thus overcoming some limitations of available experimental methods.

Many protein-protein interactions involve the binding of short protein segments to peptide-binding domains. Usually, such interactions require the ...

Assembly and Purification of Prototype Foamy Virus Intasomes

A defining feature and necessary step of the retrovirus life cycle is the integration of the viral genome into the host genome. All retroviruses encode an integrase (IN) enzyme that catalyzes the covalent joining of viral to host DNA, which is known as strand transfer. Integration may be modeled in vitro with recombinant retroviral IN and DNA oligomers mimicking the ends of the viral genome. In order to more closely recapitulate the integration reaction that occurs in vivo, integration complexes are assembled from recombinant IN and synthetic oligomers by dialysis in a reduced salt concentration buffer. The integration complex, called an intasome, may be purified by size exclusion chromatography. In the case of prototype foamy virus (PFV), the intasome is a tetramer of IN and two DNA oligomers and is readily separated from monomeric IN and free oligomer DNA. The integration efficiency of PFV intasomes may be assayed under a variety of experimental conditions to better understand the dynamics and mechanics of retroviral integration.

Recombinant retroviral integrase and DNA oligomers mimicking viral DNA ends can form an enzymatically active complex known as an intasome. Intasomes may be used for biochemical, structural, and kinetic studies. This protocol details how to assemble and purify prototype foamy virus intasomes.

A defining feature and necessary step of the retrovirus life cycle is the integration of the viral genome into the host genome. All retroviruses encode an ...

Electrophoretic Mobility Shift Assay (EMSA) for the Study of RNA-Protein Interactions: The IRE/IRP Example

RNA/protein interactions are critical for post-transcriptional regulatory pathways. Among the best-characterized cytosolic RNA-binding proteins are iron regulatory proteins, IRP1 and IRP2. They bind to iron responsive elements (IREs) within the untranslated regions (UTRs) of several target mRNAs, thereby controlling the mRNAs translation or stability. IRE/IRP interactions have been widely studied by EMSA. Here, we describe the EMSA protocol for analyzing the IRE-binding activity of IRP1 and IRP2, which can be generalized to assess the activity of other RNA-binding proteins as well. A crude protein lysate containing an RNA-binding protein, or a purified preparation of this protein, is incubated with an excess of32 P-labeled RNA probe, allowing for complex formation. Heparin is added to preclude non-specific protein to probe binding. Subsequently, the mixture is analyzed by non-denaturing electrophoresis on a polyacrylamide gel. The free probe migrates fast, while the RNA/protein complex exhibits retarded mobility; hence, the procedure is also called &#8220;gel retardation&#8221; or &#8220;bandshift&#8221; assay. After completion of the electrophoresis, the gel is dried and RNA/protein complexes, as well as free probe, are detected by autoradiography. The overall goal of the protocol is to detect and quantify IRE/IRP and other RNA/protein interactions. Moreover, EMSA can also be used to determine specificity, binding affinity, and stoichiometry of the RNA/protein interaction under investigation.

Electrophoretic Mobility Shift Assay EMSA for the Study of RNA-Protein Interactions: The IRE/IRP Example

Here we present a protocol to analyze RNA/protein interactions. The electrophoretic mobility shift assay (EMSA) is based on the differential migration of RNA/protein complexes and free RNA during native gel electrophoresis. By using a radiolabeled RNA probe, RNA/protein complexes can be visualized by autoradiography.

RNA/protein interactions are critical for post-transcriptional regulatory pathways. Among the best-characterized cytosolic RNA-binding proteins are iron ...

Biology

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.

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. ...

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ryanodine receptor (RyR) is necessary for the formation of a functional Ca2+ release channel pore. Here, we describe detailed protocols for the assessment of protein self-association, including yeast two-hybrid (Y2H), co-immunoprecipitation (co-IP) and chemical cross-linking assays. In the Y2H system, protein self-interaction is detected by &#946;-galactosidase assay in yeast co-expressing GAL4 bait and target fusions of the test protein. Protein self-interaction is further assessed by co-IP using HA- and cMyc-tagged fusions of the test protein co-expressed in mammalian HEK293 cells. The precise stoichiometry of the protein homo-oligomer is examined by cross-linking and SDS-PAGE analysis following expression in HEK293 cells. Using these different but complementary techniques, we have consistently observed the self-association of the RyR N-terminal domain and demonstrated its intrinsic ability to form tetramers. These methods can be applied to protein-protein interaction and homo-oligomerization studies of other mammalian integral membrane proteins.

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization of the ryanodine receptor, a homo-tetrameric ion channel mediating Ca2+ release from intracellular stores, is critical for skeletal and cardiac muscle contraction. Here, we present complementary in vivo and in vitro methods to detect protein self-association and determine homo-oligomer stoichiometry.

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ...

Protein Interaction Analysis in HEK-293 Cells Using Co-Immunoprecipitation

Immunoprecipitation with an Anti-Epitope Tag Affinity Gel to Study Protein-Protein Interactions

Protein-protein interactions (PPIs) play a pivotal role in biological phenomena, such as cellular organization, intracellular signal transduction, and transcriptional regulation. Therefore, understanding PPIs is an important starting point for further investigation of the function of the target protein. In this study, we propose a simple method to determine the binding of two target proteins by introducing mammalian expression vectors into HEK-293 cells using the polyethylenimine method, lysing the cells in homemade protein lysis buffer, and pulling down the target proteins on an epitope tag affinity gel. In addition, the PPI between the various epitope tag fused proteins can be confirmed by using affinity antibodies against each tag instead of the epitope tag affinity gel. This protocol could also be used to verify various PPIs, including nuclear extracts, from other cell lines. Therefore, it can be used as a basic method in a variety of PPI experiments. Proteins degrade by extended time course and repeated freeze-thaw cycles. Therefore, cell lysis, immunoprecipitation, and immunoblotting should be performed as seamlessly as possible.

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Protein-protein interactions are important for elucidating the function of target proteins, and co-immunoprecipitation (co-IP) can easily confirm PPIs. We transiently transfected a plasmid encoding an epitope-tagged protein into HEK-293 cells and developed an immunoprecipitation method to easily confirm the binding of two target proteins.

Protein-protein interactions (PPIs) play a pivotal role in biological phenomena, such as cellular organization, intracellular signal transduction, and ...

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Immunoprecipitation with an Anti-Epitope Tag Affinity Gel to Study Protein-Protein Interactions