The authors have no acknowledgements.

1. Western Blotting of CNTFR&#945; Lysates
<ol>
	<li>Express full-length sorLA and Myc-tagged CNTFR&#945; constructs in human embryonic kidney 293 (HEK293) cells using pcDNA3.1/zeo(-) and pcDNA3.1/hyg(-), respectively. Transfect the HEK293 cells with the constructs using a commercial transfection reagent according to manufacturer&#180;s protocol. Select stable clones in medium containing 150 &#181;g/mL Zeocin and/or 500 &#181;g/mL Hygromycin B Gold13.</li>
	<li>Seed equal numbers of HEK293 transfectants expressing either CNTFR&#945;-Myc or co-expressing CNTFR&#945;-Myc/sorLA in two 4-well plates as shown in Figure 1. Culture the cells in Dulbecco Modified Eagle&#180;s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 &#956;g/mL streptomycin (full medium) in a 5% carbon dioxide incubator at 37 &#730;C.</li>
	<li>Wait until the cells are 50 - 80% confluent.</li>
	<li>Remove the medium from the wells, wash once with Dulbecco Phosphate Buffered Saline (DPBS), and add fresh full medium with or without (control) 10 nM CLC:CLF-1 as indicated in Figure 1. Incubate the cells at 37 &#730;C.</li>
	<li>After 0 and 5 h of incubation, recover the medium and store it at 4 &#730;C in 1.5 mL tubes. Then add 1% Triton X-100 lysis buffer (20 mM Tris-HCl, 10 mM EDTA, pH 8.0) supplemented with a proteinase inhibitor cocktail to each well (1 min, 4 &#730;C). Subsequently, transfer the cell lysates to 1.5 mL tubes (4 &#730;C).</li>
	<li>Spin the tubes containing the medium and cell lysate (3,000 x g, 3 min, 4 &#730;C) and transfer the supernatants to 1.5 mL tubes (4 &#730;C). Discard the pellet. Transfer 5 &#956;L of each cell lysate supernatant to new 1.5 mL tubes &#8212; use them to measure the protein content later (step 1.7). Immediately, add nonreducing LDS sample buffer to all the supernatants and vortex the samples.</li>
	<li>Use the 5 &#956;L aliquots to measure the protein content in the cell lysate supernatants using a commercial protein assay according to manufacturer&#39;s protocol.</li>
	<li>Heat the samples at 95 &#730;C for 5 min.</li>
	<li>Subject an equal amount of protein from each sample to reducing SDS-PAGE13 and Western blotting13 and visualize the content of sorLA, &#946;-actin, and CNTFR&#945;-Myc in the medium and in the cell lysates using rabbit anti-sorLA16 (5 &#956;g/mL), mouse anti-&#946;-actin (0.4 &#956;g/mL), and mouse anti-Myc (1 &#956;g/mL) antibodies and the appropriate HRP-linked secondary antibodies (1:2,000).</li>
	<li>Quantify the bands by densitometry according to manufacturer&#39;s protocol.</li>
</ol>
2. Immunocytochemistry in Combination with Lysosomal Inhibitors
<ol>
	<li>Seed HEK293 cells stably transfected with CNTFR&#945;-Myc/sorLA on cover slides in a 4-well plate as shown in Figure 2. Culture the cells in full medium as above.</li>
	<li>Wait until cell confluence is 20 - 40%.</li>
	<li>Remove the medium from the wells and add full medium with or without 50 &#956;g/mL of leupeptin and of pepstatin A (leu/pep) (inhibits lysosomal hydrolases) to the cells as indicated in Figure 2. Incubate the cells at 37 &#730;C. The medium must be replaced every 6 h during the following 24 h of incubation.</li>
	<li>After 24 h of incubation, once again replace the medium (with or without leu/pep) and this time add 10 nM CLC:CLF-1. Incubate the cells at 37 &#730;C for 5 h.</li>
	<li>Wash the cells once in PBS and fix them in 4% paraformaldehyde, pH 7 for 15 min at RT. 
	Caution: Paraformaldehyde is highly toxic, avoid contact with skin, eyes, and mucous. Gloves and safety glasses should be worn and solutions made inside a fume hood.</li>
	<li>Wash the cells once in PBS and permeabilize the cells for 5 min at RT in saponin diluted in PBS (0.5% saponin).</li>
	<li>Dilute goat anti-CNTFR&#945; (1 &#956;g/mL) (notably not the mouse anti-Myc used for Western blotting) and mouse anti-LAMP-1 (15 &#956;g/mL) antibodies in 0.5% saponin and incubate the cells with the antibodies for 2 h at RT.</li>
	<li>Wash the cells 4x (5 min) in 0.5% saponin and incubate the cells with donkey anti-goat Alexa 488- and donkey anti-mouse Alexa 568-conjugated secondary antibodies (6 &#956;g/mL) for 2 h at RT.</li>
	<li>Wash the cells 4x (5 min) in 0.5% saponin, twice in deionized water (1 min), and mount the cover slides on microscope slides using mounting media.</li>
	<li>Analyze the antibody-staining of the cells by confocal microscopy using a laser-scanning confocal microscope with a 63X C-apochromat water immersion objective, NA 1.2.</li>
</ol>
3. Western Blot-detection of the pSTAT3 Level
<ol>
	<li>Seed HEK293-sorLA cells (which express endogenous levels of CNTFR&#945;) in a 4-well plate as shown in Figure 3. Culture the cells in full medium as above.</li>
	<li>Wait until the cells are 50 - 80% confluent.</li>
	<li>Remove the medium from the wells, wash once in PBS, and add fresh full medium with or without 10 nM CLC:CLF-1 to the cells as indicated in Figure 3. Incubate the cells at 37 &#730;C for 5 h.</li>
	<li>Remove the medium, wash the cells twice in unsupplemented DMEM (blank medium; without FBS and antibiotics), and then reincubate the cells for 90 min (37 &#730;C) in blank medium.</li>
	<li>Once again, remove the medium and add fresh blank medium with or without 5 nM CLC:CLF-1 (to initiate STAT3 phosphorylation) as indicated in Figure 3. Incubate for 15 min at 37 &#730;C.</li>
	<li>Quickly remove the medium and lyse the cells (1 min, 4 &#730;C) by adding 1% Triton X-100 lysis buffer (20 mM Tris-HCl, 10 mM EDTA, pH 8.0) supplemented with a proteinase inhibitor cocktail and a phosphatase inhibitor cocktail to each well. Subsequently, transfer the cell lysates to 1.5 mL tubes (4 &#730;C).</li>
	<li>Transfer 5 &#956;L of each supernatant to new 1.5 mL tubes &#8212; use them to measure the protein content later (step 3.8). Immediately, add nonreducing LDS sample buffer to all the supernatants and vortex the samples.</li>
	<li>Measure the protein content of the 5 &#956;L aliquots using a commercial protein assay according to manufacturer&#39;s protocol.</li>
	<li>Sonicate each sample at RT until&#160;they are no longer viscous (approximately 1 min) and subsequently heat them at 95 &#730;C for 5 min.</li>
	<li>Subject an equal amount of protein from each sample to SDS-PAGE13 and Western blotting13 and visualize the content of pSTAT3 and total STAT3 in the cell lysates using rabbit anti-pSTAT3 (1:2000) and mouse anti-STAT3 (1:1,000) antibodies and the appropriate HRP-linked mouse and rabbit secondary antibodies (1:2,000).</li>
	<li>Quantify the bands by densitometry according to manufacturer&#39;s protocol.</li>
</ol>

<ol>
	<li>Elson, G. C., 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=CLF+associates+with+CLC+to+form+a+functional+heteromeric+ligand+for+the+CNTF+receptor+complex.">CLF associates with CLC to form a functional heteromeric ligand for the CNTF receptor complex.</a> Nat Neurosci. 3 (9), 867-872 (2000).</li><li>Senaldi, G., 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=Novel+neurotrophin-1/B+cell-stimulating+factor-3:+a+cytokine+of+the+IL-6+family.">Novel neurotrophin-1/B cell-stimulating factor-3: a cytokine of the IL-6 family.</a> Proc Natl Acad Sci U S A. 96 (20), 11458-11463 (1999).</li><li>Elson, G. C., 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=Cytokine-like+factor-1,+a+novel+soluble+protein,+shares+homology+with+members+of+the+cytokine+type+I+receptor+family.">Cytokine-like factor-1, a novel soluble protein, shares homology with members of the cytokine type I receptor family.</a> J Immunol. 161 (3), 1371-1379 (1998).</li><li>Lelievre, E., 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=Signaling+pathways+recruited+by+the+cardiotrophin-like+cytokine/cytokine-like+factor-1+composite+cytokine:+specific+requirement+of+the+membrane-bound+form+of+ciliary+neurotrophic+factor+receptor+alpha+component.">Signaling pathways recruited by the cardiotrophin-like cytokine/cytokine-like factor-1 composite cytokine: specific requirement of the membrane-bound form of ciliary neurotrophic factor receptor alpha component.</a> J Biol Chem. 276 (25), 22476-22484 (2001).</li><li>Zou, X., 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=Neonatal+Death+in+Mice+Lacking+Cardiotrophin-Like+Cytokine+(CLC)+is+Associated+with+Multifocal+Neuronal+Hypoplasia.">Neonatal Death in Mice Lacking Cardiotrophin-Like Cytokine (CLC) is Associated with Multifocal Neuronal Hypoplasia.</a> Vet Pathol. 46 (3), 514-519 (2008).</li><li>Forger, N. G., 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=Cardiotrophin-like+cytokine/cytokine-like+factor+1+is+an+essential+trophic+factor+for+lumbar+and+facial+motoneurons+in+vivo.">Cardiotrophin-like cytokine/cytokine-like factor 1 is an essential trophic factor for lumbar and facial motoneurons in vivo.</a> J Neurosci. 23 (26), 8854-8858 (2003).</li><li>Alexander, W. 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=Suckling+defect+in+mice+lacking+the+soluble+haemopoietin+receptor+NR6.">Suckling defect in mice lacking the soluble haemopoietin receptor NR6.</a> Curr Biol. 9 (11), 605-608 (1999).</li><li>DeChiara, T. M., 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=Mice+lacking+the+CNTF+receptor,+unlike+mice+lacking+CNTF,+exhibit+profound+motor+neuron+deficits+at+birth.">Mice lacking the CNTF receptor, unlike mice lacking CNTF, exhibit profound motor neuron deficits at birth.</a> Cell. 83 (2), 313-322 (1995).</li><li>Crisponi, L., 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=Crisponi+syndrome+is+caused+by+mutations+in+the+CRLF1+gene+and+is+allelic+to+cold-induced+sweating+syndrome+type+1.">Crisponi syndrome is caused by mutations in the CRLF1 gene and is allelic to cold-induced sweating syndrome type 1.</a> Am J Hum Genet. 80 (5), 971-981 (2007).</li><li>Crisponi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autosomal+recessive+disorder+with+muscle+contractions+resembling+neonatal+tetanus,+characteristic+face,+camptodactyly,+hyperthermia,+and+sudden+death:+a+new+syndrome.">Autosomal recessive disorder with muscle contractions resembling neonatal tetanus, characteristic face, camptodactyly, hyperthermia, and sudden death: a new syndrome.</a> Am J Med Genet. 62 (4), 365-371 (1996).</li><li>Sohar, E., Shoenfeld, Y., Udassin, R., Magazanik, A., Revach, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cold-induced+profuse+sweating+on+back+and+chest.+A+new+genetic+entity.">Cold-induced profuse sweating on back and chest. A new genetic entity.</a> Lancet. 2 (8099), 1073-1074 (1978).</li><li>Guillet, C., 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=Functionally+active+fusion+protein+of+the+novel+composite+cytokine+CLC/soluble+CNTF+receptor.">Functionally active fusion protein of the novel composite cytokine CLC/soluble CNTF receptor.</a> Eur J Biochem. 269 (7), 1932-1941 (2002).</li><li>Larsen, J. V., 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=Cytokine-Like+Factor+1,+an+Essential+Facilitator+of+Cardiotrophin-Like+Cytokine:Ciliary+Neurotrophic+Factor+Receptor+alpha+Signaling+and+sorLA-Mediated+Turnover.">Cytokine-Like Factor 1, an Essential Facilitator of Cardiotrophin-Like Cytokine:Ciliary Neurotrophic Factor Receptor alpha Signaling and sorLA-Mediated Turnover.</a> Mol Cell Biol. 36 (8), 1272-1286 (2016).</li><li>Willnow, T. E., Petersen, C. M., Nykjaer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VPS10P-domain+receptors+-+regulators+of+neuronal+viability+and+function.">VPS10P-domain receptors - regulators of neuronal viability and function.</a> Nat Rev Neurosci. 9 (12), 899-909 (2008).</li><li>Jacobsen, L., 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=Molecular+characterization+of+a+novel+human+hybrid-type+receptor+that+binds+the+alpha2-macroglobulin+receptor-associated+protein.">Molecular characterization of a novel human hybrid-type receptor that binds the alpha2-macroglobulin receptor-associated protein.</a> J Biol Chem. 271 (49), 31379-31383 (1996).</li><li>Nielsen, M. 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=Sorting+by+the+cytoplasmic+domain+of+the+amyloid+precursor+protein+binding+receptor+SorLA.">Sorting by the cytoplasmic domain of the amyloid precursor protein binding receptor SorLA.</a> Mol Cell Biol. 27 (19), 6842-6851 (2007).</li><li>Jacobsen, L., 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=The+sorLA+cytoplasmic+domain+interacts+with+GGA1+and+-2+and+defines+minimum+requirements+for+GGA+binding.">The sorLA cytoplasmic domain interacts with GGA1 and -2 and defines minimum requirements for GGA binding.</a> FEBS Lett. 511 (1-3), 155-158 (2002).</li><li>Klinger, S. C., 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=SorLA+regulates+the+activity+of+lipoprotein+lipase+by+intracellular+trafficking.">SorLA regulates the activity of lipoprotein lipase by intracellular trafficking.</a> J Cell Sci. 124 (7), 1095-1105 (2011).</li><li>Kitago, Y., 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=Structural+basis+for+amyloidogenic+peptide+recognition+by+sorLA.">Structural basis for amyloidogenic peptide recognition by sorLA.</a> Nat Struct Mol Biol. 22 (3), 199-206 (2015).</li><li>Quistgaard, E. M., 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=Ligands+bind+to+Sortilin+in+the+tunnel+of+a+ten-bladed+beta-propeller+domain.">Ligands bind to Sortilin in the tunnel of a ten-bladed beta-propeller domain.</a> Nat Struct Mol Biol. 16 (1), 96-98 (2009).</li><li>Larsen, J. V., 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=Human+sorCS1+binds+sortilin+and+hampers+its+cellular+functions.">Human sorCS1 binds sortilin and hampers its cellular functions.</a> Biochem J. 457 (2), 277-288 (2014).</li><li>Larsen, J. V., 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=Sortilin+facilitates+signaling+of+ciliary+neurotrophic+factor+and+related+helical+type+1+cytokines+targeting+the+gp130/leukemia+inhibitory+factor+receptor+beta+heterodimer.">Sortilin facilitates signaling of ciliary neurotrophic factor and related helical type 1 cytokines targeting the gp130/leukemia inhibitory factor receptor beta heterodimer.</a> Mol Cell Biol. 30 (17), 4175-4187 (2010).</li></ol>

The authors have nothing to disclose.

The protocol described here can be used specifically to demonstrate the sorLA-mediated CLC:CLF-1-dependent downregulation and lysosomal targeting of CNTFR&#945;, as well as the accompanying weakened response to CLC:CLF-1 stimulation.
The HEK293 cell line is well-suited for this protocol, as they have only a minor endogenous expression of CNTFR&#945; and sorLA, are easy to transfect, express gp130/LIFR&#946;, and have a large cell body, which is suited for immunocytochemistry. However, in theory any cell line with similar properties should work as well.
The protocol has two critical steps. The first concerns step 2.3, in which the leu/pep medium must be replaced approximately every 6 h for 24 h. Failure to do so may result in active lysosomal enzymes and less or no detectable accumulation of protein in the lysosomes. The second critical step (3.4) concerns washing upon pre-incubation with CLC:CLF-1. It is important to remove any unbound ligand, and to allow the cells time to recover (avoiding continued stimulation) in unsupplemented DMEM (blank medium). This will ensure a low background level of pSTAT3 during the subsequent re-stimulation with CLC:CLF-1 (step 3.5).
Notably, Figure 6 demonstrates that the cellular response (pSTAT3) decreases in parallel with the sorLA-mediated downregulation of CNTFR&#945;. It should be emphasized however, that although a reduced response is in accordance with (and to be expected upon) a reduction of the CNTFR&#945; pool, it does not prove that the low CNTFR&#945; expression alone accounts for the reduced cellular response. Thus, changes &#8212; resulting from pre-stimulation or transfection &#8212; in any of the functional elements of the signaling pathway upstream to pSTAT3 may have contributed to the altered response.
The basic concept of the protocol is not limited to this particular receptor system. By modifying the protocol (i.e. another cell line, other antibodies, other ligands, etc.) it can be used to determine the ligand-induced downregulation of other receptors as well &#8212; regardless of sorLA&#180;s involvement. However, it should be noted that analysis of receptor-downregulation by Western blotting is mainly suited for receptors, e.g., GPI-anchored receptors, which exhibit a slow turnover and a low degree of new synthesis in unstimulated cells. Thus, in receptors showing a high level of new-synthesis, ligand-induced downregulation may be compensated for by synthesis of new receptors. Under these circumstances, the downregulation of the receptor pool can instead be determined by means of metabolic labeling and pulse-chase experiments as described in21. Alternatively, iodinated antibodies (preferably Fc-fragments) that do not interfere with receptor binding can be used to &#34;tag&#34; the ectodomain of the receptor, and the expected downregulation can then be determined by radioactive counting of cell pellet and medium.
For logistic reasons we transfected our cells with a CNTFR&#945; construct carrying a Myc-tag, and in the present protocol a mouse anti-Myc antibody was used for Western blot-detection of the receptor. In contrast, a goat anti-CNTFR&#945; antibody was used for immunocytochemistry and double labeling as LAMP-1 was detected by a mouse antibody &#8212; and obviously use of two primary mouse antibodies would result in unspecific (overlapping) staining with secondary antibodies. Note that since the goat anti-CNTFR&#945; also works in Western blotting (not shown), the protocol could easily be modified and applied to cells transfected with untagged CNTFR&#945;.
Finally, it has recently been shown that also the endocytic receptor sortilin binds CLC:CLF-1 with high affinity22. Thus, it is conceivable that sortilin, like sorLA, interconnects to CLC:CNTFR&#945; via CLF-1 and is able to mediate endocytosis and lysosomal targeting of CNTFR&#945; as well. Using sortilin instead of sorLA, the present protocol may be used to clarify this question.

The CLC and&#160;CLF-1 constitute the two subunits of the heterodimeric cytokine CLC:CLF-11-3. For cellular signal induction, CLC:CLF-1 first targets the CNTFR&#945;, its primary and GPI-anchored receptor, to form a membrane-bound trimeric complex. The CLC:CLF-1:CNTFR&#945; complex subsequently recruits gp130/LIFR&#946; which mediates transmembrane signaling via the Janus Kinase/Signal Transducers and Activators of Transcription 3 (JAK/STAT3) pathway4. Mice deficient in any of the three subunits display one and the same phenotype and die within 24 h after birth due to a reduced number of facial neurons and insufficient suckling5-8. Findings in humans likewise underscore the functional coherence of the three subunits. Thus, human mutations (homozygote or compound) causing dysfunction of CLC:CLF-1 or CNTFR&#945;, all result in the so-called cold-induced sweating/Crisponi syndrome, a condition characterized by symptoms such as impaired suckling and swallowing, various dysmorphic features, temperature spikes, and paradoxical and perfuse sweating at low temperatures9-11.
In vitro, the combination of CLC and CNTFR&#945; is both necessary and sufficient for interaction with gp130/LIFR&#946; and the induction of signaling in cells12. The role of CLF-1 on the other hand is less clear. It is not directly involved in signaling, and has long been regarded as an appendix that mainly serves to facilitate the cellular secretion of CLC1. However, recent findings show that CLF-1 has additional and more important functions implicating both the signaling and turnover of CLC and CNTFR&#945;. Thus, it appears that CLF-1 contains three independent binding sites: one for its well-known binding to CLC; one that mediates direct binding to the CNTFR&#945;; and a third (high affinity) site for interaction with the endocytic receptor sorLA. As both CLC and CLF-1 seem&#160;to target CNTFR&#945; with a considerable lower affinity than the CLC:CLF-1 complex, it is conceivable that CLF-1 (via its CNTFR&#945;-binding site) promotes the unification of CLC and CNTFR&#945; and thereby facilitates signaling13.
CLF-1&#39;s interaction with sorLA, the main issue of the present presentation, plays a completely different role. SorLA is one of the five type 1 receptors that constitute the Vps10p-domain receptor family14. It is expressed in a variety of tissues but in particular in brain and neuronal tissues15. Similar to the other family members sorLA carries an N-terminal ligand binding Vps10p-domain, but in addition, it also comprises other domain types including ligand-binding elements found in members of the low-density lipoprotein receptor family15. Its cytoplasmic tail interacts with several adaptor proteins, e.g., adaptor protein-1 and -2, and sorLA conveys efficient endocytosis as well as intracellular sorting and transport of bound proteins16-18. The Vps10p-domain comprises a large ten-bladed &#946;-propeller19,20, which binds a series of unrelated ligands including CLF-1 (but notably, not CLC)13. The binding site in CLF-1 is accessible even after complex formation with CLC and CNTFR&#945; which means that sorLA binds not only free CLF-1, but also CLC:CLF-1 and the trimeric complex CLC:CLF-1:CNTFR&#945;13. SorLA conveys rapid endocytosis of CLF-1 and CLC:CLF-1, but these are soluble proteins whereas CNTFR&#945; is fixed to the surface membrane by a GPI-anchor. The question is therefore if binding of CLC:CLF-1:CNTFR&#945; allows sorLA to internalize the entire complex and thereby to alter the surface-membrane expression of CNTFR&#945; (and the subsequent cellular susceptibility to CLC:CLF-1 signal induction), and/or the turnover of CNTFR&#945;.
The experiments described in the present report were designed to clarify the following questions:
Does sorLA mediate CLC:CLF-1-dependent downregulation of surface-membrane CNTFR&#945;? To clarify this, it was initially tried to determine the turnover of CNTFR&#945; (in the absence and presence of sorLA) by means of metabolic labeling and pulse-chase experiments as described in21. However, attempts to label CNTFR&#945; using a mixture of [S35] cysteine and [S35] methionine showed poor incorporation of radioactivity. This suggested a very low degree of new-synthesis, which in all probability would be unable to compensate for a sudden and significant loss of receptors. To determine if CNTFR&#945; was downregulated when interconnected via CLC:CLF-1 to sorLA, it was therefore decided simply to measure (using Western blotting) the total cellular pool of CNTFR&#945; before and after exposure to CLC:CLF-1 - and to compare results in cells transfected or not transfected with sorLA.
Does sorLA target CNTFR&#945; to lysosomes? To answer this question, the localization of CNTFR&#945; &#8212; internalized via its CLC:CLF-1-mediated binding to sorLA &#8212; was examined using immunocytochemistry, and labeling with antibodies directed towards CNTFR&#945; and the late-endosome/lysosome marker Lysosomal-associated Membrane Protein 1 (LAMP-1). The experiment was performed on cells treated as well as untreated with inhibitors of lysosomal enzymes. The idea was of course that if CNTFR&#945; was degraded in lysosomes, cells treated with enzyme-inhibitors would present CNTFR&#945;-staining accumulating in LAMP-1 positive vesicles, whereas untreated cells would show little or no staining for CNTFR&#945;.
Does CNTFR&#945; downregulation lower the cellular response to CLC:CLF-1 stimulation? The trimeric complex consisting of CLC, CLF-1, and CNTFR&#945; signals via the gp130/LIFR&#946; heterodimer using the JAK/STAT3 pathway. Accordingly, the capacity of cells to respond to CLC:CLF-1 signaling upon downregulation of CNTFR&#945; was explored by Western blot detection and quantitation of the phospho-STAT3 (pSTAT3)/total STAT3 level in sorLA transfectants.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Zeocin</td>
			<td>InvivoGen</td>
			<td>ant-zn-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Hygromycin B Gold</td>
			<td>InvivoGen</td>
			<td>ant-hg-1</td>
			<td></td>
		</tr>
		<tr>
			<td>FuGENE 6 Transfection Reagent&#160;</td>
			<td>Roche</td>
			<td>11 815 091 001</td>
			<td></td>
		</tr>
		<tr>
			<td>4-well plates (Nunclon Delta Surface)</td>
			<td>Thermo Scientific</td>
			<td>176740</td>
			<td></td>
		</tr>
		<tr>
			<td>Safe-Lock tubes</td>
			<td>Eppendorf</td>
			<td>0030 120.086</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco Modified Eagle&#180;s Medium</td>
			<td>Lonza</td>
			<td>BE12-604F/U1</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Thermo Scientific</td>
			<td>10270106</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermo Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco Phosphate Buffered Saline&#160;</td>
			<td>HyClone</td>
			<td>SH30028.02</td>
			<td></td>
		</tr>
		<tr>
			<td>CLC:CLF-1</td>
			<td>R&#38;D Systems</td>
			<td>1151-CL/CF</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-LAMP-1&#160;</td>
			<td>DSHP</td>
			<td>H4A3</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-sorLA</td>
			<td></td>
			<td></td>
			<td>Reference 16</td>
		</tr>
		<tr>
			<td>Mouse anti-b-actin&#160;</td>
			<td>Sigma</td>
			<td>A2228</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-Myc</td>
			<td>Genscript</td>
			<td>A00704</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-CNTFRa&#160;</td>
			<td>R&#38;D Systems</td>
			<td>AF-303</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-pSTAT3&#160;</td>
			<td>Cell signaling Technology</td>
			<td>9145s</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-STAT3&#160;</td>
			<td>Cell signaling Technology</td>
			<td>9139s</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse HRP-linked antibody</td>
			<td>Cell signaling Technology</td>
			<td>7076s</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-rabbit HRP-linked antibody</td>
			<td>Cell signaling Technology</td>
			<td>7074s</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-goat HRP-linked antibody</td>
			<td>Dako</td>
			<td>P0160</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-goat secondary antibody, Alexa Fluor 488 conjugate</td>
			<td>Invitrogen</td>
			<td>A11055</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-mouse secondary antibody, Alexa Fluor 568 conjugate</td>
			<td>Invitrogen</td>
			<td>A10037</td>
			<td></td>
		</tr>
		<tr>
			<td>Leupeptin</td>
			<td>Sigma</td>
			<td>L8511-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Pepstatin A&#160;</td>
			<td>Sigma</td>
			<td>P4265-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100&#160;</td>
			<td>AppliChem</td>
			<td>A1388</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Merck</td>
			<td>1,082,191,000</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA:Titriplex III</td>
			<td>Merck</td>
			<td>1,084,180,250</td>
			<td></td>
		</tr>
		<tr>
			<td>cOmplete mini EDTA-free</td>
			<td>Roche</td>
			<td>11836170001</td>
			<td></td>
		</tr>
		<tr>
			<td>PhosSTOP</td>
			<td>Roche</td>
			<td>04 906 837 001</td>
			<td></td>
		</tr>
		<tr>
			<td>LDS sample Buffer (4X)&#160;</td>
			<td>Novex</td>
			<td>NP0007</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-Rad Protein Assay</td>
			<td>Bio-Rad</td>
			<td>500-0006</td>
			<td></td>
		</tr>
		<tr>
			<td>4% paraformaldehyde</td>
			<td>VWR</td>
			<td>97,135,000</td>
			<td></td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Sigma</td>
			<td>S7900-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting Media</td>
			<td>Dako</td>
			<td>S3023</td>
			<td></td>
		</tr>
	</tbody>
</table>

sorla and clc:clf-1-dependent downregulation of cntfr&#945; as demonstrated by western blotting, inhibition of lysosomal enzymes, and immunocytochemistry

The heterodimeric cytokine Cardiotrophin-like Cytokine:Cytokine-like Factor-1 (CLC:CLF-1) targets the glycosylphosphatidylinositol (GPI)-anchored CNTFR&#945; to form a trimeric complex that subsequently recruits glycoprotein 130/Leukemia Inhibitory Factor Receptor-&#946; (gp130/LIFR&#946;) for signaling. Both CLC and CNTFR&#945; are necessary for signaling but so far CLF-1 has only been known as a putative facilitator of CLC secretion. However, it has recently been shown that CLF-1 contains three binding sites: one for CLC; one for CNTFR&#945; (that may promote assembly of the trimeric complex); and one for the endocytic receptor sorLA. The latter site provides high affinity binding of CLF-1, CLC:CLF-1, as well as the trimeric (CLC:CLF-1:CNTFR&#945;) complex to sorLA, and in sorLA-expressing cells the soluble ligands CLF-1 and CLC:CLF-1 are rapidly taken up and internalized. In cells co-expressing CNTFR&#945; and sorLA, CNTFR&#945; first binds CLC:CLF-1 to form a membrane-associated trimeric complex, but it also connects to sorLA via the free sorLA-binding site in CLF-1. As a result, CNTFR&#945;, which has no capacity for endocytosis on its own, is tugged along and internalized by the sorLA-mediated endocytosis of CLC:CLF-1.
The present protocol describes the experimental procedures used to demonstrate i) the sorLA-mediated and CLC:CLF-1-dependent downregulation of surface-membrane CNTFR&#945; expression; ii) sorLA-mediated endocytosis and lysosomal targeting of CNTFR&#945;; and iii) the lowered cellular response to CLC:CLF-1-stimulation upon sorLA-mediated downregulation of CNTFR&#945;.

Figure 1 shows a schematic representation of the cell-seeding and CLC:CLF-1 incubation in protocol 1. Figure 2 shows a schematic representation of the cell-seeding and leu/pep and CLC:CLF-1 incubation in protocol 2. Figure 3 shows a schematic representation of the cell-seeding and CLC:CLF-1 incubation and stimulation in protocol 3. Figure 4 shows the sorLA-mediated CLC:CLF-1-dependent downregulation of CNTFR&#945;. Figure 5 shows the sorLA-mediated endocytosis and lysosomal targeting of CNTFR&#945;. Figure 6 shows the lower response to CLC:CLF-1 stimulation after CNTFR&#945; downregulation.
Note that bands signifying CNTFR&#945;-Myc have similar density at time zero, whereas downregulation of CNTFR&#945;-Myc is demonstrated by the weaker band in sorLA transfectants after 5 h (Figure 4). Figure 5 shows that CNTFR&#945;-Myc has accumulated in LAMP-1 positive vesicles of leu/pep treated cells. In contrast, no accumulation of CNTFR&#945;-Myc is seen in cells not subjected to leu/pep treatment. This demonstrates that CNTFR&#945;-Myc is sorted to lysosomes and degraded. As can be seen in Figure 6, the level of pSTAT3 is significantly reduced in pre-stimulated cells as compared to the level in cells that has not been exposed to CLC:CLF-1. This is in accordance with the observed sorLA-mediated downregulation of CNTFR&#945; in the pre-stimulated cells (Figure 4).
<img alt="Figure 1" src="/files/ftp_upload/55019/55019fig1.jpg" /> 
Figure 1: Schematic representation of cell-seeding and CLC:CLF-1 incubation in protocol 1. Seed HEK293-CNTFR&#945;-Myc and HEK293-CNTFR&#945;-Myc/sorLA cells in two 4-well plates (0 and 5 h) and incubate cells with CLC:CLF-1 as indicated. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55019/55019fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55019/55019fig2.jpg" /> 
Figure 2: Schematic representation of cell-seeding and leu/pep and CLC:CLF-1 incubation in protocol 2. Seed HEK293-CNTFR&#945;-Myc/sorLA cells on cover slides in a&#160;4-well plate and incubate cells with or without leu/pep and CLC:CLF-1 as indicated. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55019/55019fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55019/55019fig3.jpg" /> 
Figure 3: Schematic representation of cell-seeding and CLC:CLF incubation and stimulation in protocol 3. Seed HEK293-sorLA cells in one 4-well plate and incubate cells with or without CLC:CLF-1 (pre-exp.) to downregulate the CNTFR&#945; followed by CLC:CLF-1 stimulation (stim.) to initiate STAT3 phosphorylation as indicated. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55019/55019fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/55019/55019fig4.jpg" /> 
Figure 4: Results showing that SorLA mediates CLC:CLF-1-dependent downregulation of CNTFR&#945;. (A) HEK293-CNTFR&#945;-Myc and (B) HEK293-CNTFR&#945;-Myc/sorLA cells were incubated in the absence (white columns) or presence (grey columns) of 10 nM CLC:CLF-1. After 0 and 5 h, the incubation was stopped and the content of CNTFR&#945;-Myc in the medium (m) and in the cell lysates (l) was detected by Western blotting and quantified by densitometry. The upper panels show Western blot results from representative experiments and the lower panels show the detected levels of CNTFR&#945;-Myc found in the cell lysates. The levels are shown relative to the CNTFR&#945;-Myc level in the single and double transfectants at 0 h. Each column represents mean &#177;SEM (n = 3). p-values calculated using t-test. Reproduced after original figure13.&#160;<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55019/55019fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<img alt="Figure 5" src="/files/ftp_upload/55019/55019fig5.jpg" /> 
Figure 5: Results showing that SorLA mediates endocytosis and lysosomal targeting of CNTFR&#945;. HEK293-CNTFR&#945;-Myc/sorLA cells were treated with or without leu/pep, incubated with 10 nM CLC:CLF-1 for 5 h, fixed, and finally stained using anti-CNTFR&#945; and anti-LAMP-1 antibodies as described in protocol 2. Scale bars: 5 &#956;m. Reproduced after original figure13. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55019/55019fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/55019/55019fig6.jpg" /> 
Figure 6: SorLA-mediated downregulation of CNTFR&#945; is accompanied by a lowered cellular response to CLC:CLF-1 stimulation. 
HEK293-sorLA cells were pre-incubated in the absence or presence of 10 nM CLC:CLF-1 for 5 h (pre-exp.), starved in blank medium for 90 min, and stimulated with 5 nM CLC:CLF-1 for 15 min (stim.). (left) The columns show the relative levels of pSTAT3 in the cells. Each column represents mean &#177;SEM (n = 3) relative to the pSTAT3 level in cells preincubated in the absence of CLC:CLF-1 but stimulated with CLC:CLF-1. p-value calculated using t-test. (right) The Western blot shows the response of pSTAT3 and STAT3 obtained in a representative experiment. Reproduced after original figure13. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55019/55019fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about SorLA and CLC:CLF-1-dependent Downregulation of CNTFRÎ± as Demonstrated by Western Blotting, Inhibition of Lysosomal Enzymes, and Immunocytochemistry at JoV

SorLA and CLC:CLF-1-dependent Downregulation of CNTFR&#945; as Demonstrated by Western Blotting, Inhibition of Lysosomal Enzymes, and Immunocytochemistry

The present protocol describes how Western blotting and immunocytochemistry combined with inhibition of lysosomal enzymes can be used to demonstrate the downregulation of Ciliary Neurotrophic Factor Receptor-&#945; (CNTFR&#945;), which is conveyed by the interaction between Cytokine-like Factor-1 (CLF-1) and the endocytic receptor sorLA.

The heterodimeric cytokine Cardiotrophin-like Cytokine:Cytokine-like Factor-1 (CLC:CLF-1) targets the glycosylphosphatidylinositol (GPI)-anchored ...

sorla-clcclf-1-dependent-downregulation-cntfr-as-demonstrated-western

Nanyang Technological University

Research

JoVE Journal

Biochemistry

7.2K Views.  Aarhus University. The present protocol describes how Western blotting and immunocytochemistry combined with inhibition of lysosomal enzymes can be used to demonstrate the downregulation of Ciliary Neurotrophic Factor Receptor-α (CNTFRα), which is conveyed by the interaction between Cytokine-like Factor-1 (CLF-1) and the endocytic receptor sorLA.

Video: SorLA and CLC:CLF-1-dependent Downregulation of CNTFRα as Demonstrated by Western Blotting, Inhibition of Lysosomal Enzymes, and Immunocytochemistry

High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper and food industries. A deeper understanding of CAZymes is important from both fundamental biology and industrial standpoints. Vast numbers of CAZymes exist in nature (especially in microorganisms) and hundreds of thousands have been cataloged and described in the carbohydrate active enzyme database (CAZy). However, the rate of discovery of putative enzymes has outstripped our ability to biochemically characterize their activities. One reason for this is that advances in genome and transcriptome sequencing, together with associated bioinformatics tools allow for rapid identification of candidate CAZymes, but technology for determining an enzyme's biochemical characteristics has advanced more slowly. To address this technology gap, a novel high-throughput assay kit based on insoluble chromogenic substrates is described here. Two distinct substrate types were produced: Chromogenic Polymer Hydrogel (CPH) substrates (made from purified polysaccharides and proteins) and Insoluble Chromogenic Biomass (ICB) substrates (made from complex biomass materials). Both CPH and ICB substrates are provided in a 96-well high-throughput assay system. The CPH substrates can be made in four different colors, enabling them to be mixed together and thus increasing assay throughput. The protocol describes a 96-well plate assay and illustrates how this assay can be used for screening the activities of enzymes, enzyme cocktails, and broths.

A high-throughput assay for enzyme screening is described. This multiplexed ready-to-use assay kit comprises of pre-chosen Chromogenic Polymer Hydrogel (CPH) substrates and complex Insoluble Chromogenic Biomass (ICB) substrates. Target enzymes are polysaccharide degrading endo-enzymes and proteases.

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper ...

Identification of Plant Ice-binding Proteins Through Assessment of Ice-recrystallization Inhibition and Isolation Using Ice-affinity Purification

Ice-binding proteins (IBPs) belong to a family of stress-induced proteins that are synthesized by certain organisms exposed to subzero temperatures. In plants, freeze damage occurs when extracellular ice crystals grow, resulting in the rupture of plasma membranes and possible cell death. Adsorption of IBPs to ice crystals restricts further growth by a process known as ice-recrystallization inhibition (IRI), thereby reducing cellular damage. IBPs also demonstrate the ability to depress the freezing point of a solution below the equilibrium melting point, a property known as thermal hysteresis (TH) activity. These protective properties have raised interest in the identification of novel IBPs due to their potential use in industrial, medical and agricultural applications. This paper describes the identification of plant IBPs through 1) the induction and extraction of IBPs in plant tissue, 2) the screening of extracts for IRI activity, and 3) the isolation and purification of IBPs. Following the induction of IBPs by low temperature exposure, extracts are tested for IRI activity using a &#39;splat assay&#39;, which allows the observation of ice crystal growth using a standard light microscope. This assay requires a low protein concentration and generates results that are quickly obtained and easily interpreted, providing an initial screen for ice binding activity. IBPs can then be isolated from contaminating proteins by utilizing the property of IBPs to adsorb to ice, through a technique called &#39;ice-affinity purification&#39;. Using cell lysates collected from plant extracts, an ice hemisphere can be slowly grown on a brass probe. This incorporates IBPs into the crystalline structure of the polycrystalline ice. Requiring no a priori biochemical or structural knowledge of the IBP, this method allows for recovery of active protein. Ice-purified protein fractions can be used for downstream applications including the identification of peptide sequences by mass spectrometry and the biochemical analysis of native proteins.

This paper outlines the identification of ice-binding proteins from freeze-tolerant plants through the assessment of ice-recrystallization inhibition activity and subsequent isolation of native IBPs using ice-affinity purification.

Ice-binding proteins (IBPs) belong to a family of stress-induced proteins that are synthesized by certain organisms exposed to subzero temperatures. In ...

Protein Film Infrared Electrochemistry Demonstrated for Study of H2 Oxidation by a [NiFe] Hydrogenase

Understanding the chemistry of redox proteins demands methods that provide precise control over redox centers within the protein. The technique of protein film electrochemistry, in which a protein is immobilized on an electrode surface such that the electrode replaces physiological electron donors or acceptors, has provided functional insight into the redox reactions of a range of different proteins. Full chemical understanding requires electrochemical control to be combined with other techniques that can add additional structural and mechanistic insight. Here we demonstrate a technique, protein film infrared electrochemistry, which combines protein film electrochemistry with infrared spectroscopic sampling of redox proteins. The technique uses a multiple-reflection attenuated total reflectance geometry to probe a redox protein immobilized on a high surface area carbon black electrode. Incorporation of this electrode into a flow cell allows solution pH or solute concentrations to be changed during measurements. This is particularly powerful in addressing redox enzymes, where rapid catalytic turnover can be sustained and controlled at the electrode allowing spectroscopic observation of long-lived intermediate species in the catalytic mechanism. We demonstrate the technique with experiments on E. coli hydrogenase 1 under turnover (H2 oxidation) and non-turnover conditions.

Protein Film Infrared Electrochemistry Demonstrated for Study of H2 Oxidation by a [NiFe] Hydrogenase

Here, we describe a technique, protein film infrared electrochemistry, which allows immobilized redox proteins to be studied spectroscopically under direct electrochemical control at a carbon electrode. Infrared spectra of a single protein sample can be recorded at a range of applied potentials and under a variety of solution conditions.

Understanding the chemistry of redox proteins demands methods that provide precise control over redox centers within the protein. The technique of protein ...

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from the oxidation of reduced compounds such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2). The complete annotation of the Arabidopsis thaliana genome has established it as the most widely used plant model system, and thus the need to purify mitochondria from a variety of organs (leaf, root, or flower) is necessary to fully utilize the tools that are now available for Arabidopsis to study mitochondrial biology. Mitochondria are isolated by homogenization of the tissue using a variety of approaches, followed by a series of differential centrifugation steps producing a crude mitochondrial pellet that is further purified using continuous colloidal density gradient centrifugation. The colloidal density material is subsequently removed by multiple centrifugation steps. Starting from 100 g of fresh leaf tissue, 2 - 3 mg of mitochondria can be routinely obtained. Respiratory experiments on these mitochondria display typical rates of 100 - 250 nmol O2 min-1 mg total mitochondrial protein-1 (NADH-dependent rate) with the ability to use various substrates and inhibitors to determine which substrates are being oxidized and the capacity of the alternative and cytochrome terminal oxidases. This protocol describes an isolation method of mitochondria from Arabidopsis thaliana leaves using continuous colloidal density gradients and an efficient respiratory measurements of purified plant mitochondria.

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

As mitochondria are only a small percentage of the plant cell, they need to be purified for a range of studies. Mitochondria can be isolated from a variety of plant organs by homogenization, followed by differential and density gradient centrifugation to obtain a highly purified mitochondrial fraction.

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from ...

A Western Blotting Protocol for Small Numbers of Hematopoietic Stem Cells

Hematopoietic stem cells (HSCs) are rare cells, with the mouse bone marrow containing only ~25,000 phenotypic long term repopulating HSCs. A Western blotting protocol was optimized and suitable for the analysis of small numbers of HSCs (500 - 15,000 cells). Phenotypic HSCs were purified, accurately counted, and directly lysed in Laemmli sample buffer. Lysates containing equal numbers of cells were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the blot was prepared and processed following standard Western blotting protocols. Using this protocol, 2,000 - 5,000 HSCs can be routinely analyzed, and in some cases data can be obtained from as few as 500 cells, compared to the 20,000 to 40,000 cells reported in most publications. This protocol should be generally applicable to other hematopoietic cells, and enables the routine analysis of small numbers of cells using standard laboratory procedures.

A standard Western blotting protocol was optimized for analyzing as few as 500 hematopoietic stem or progenitor cells. Optimization involves careful handling of the cell sample, limiting transfers between tubes, and directly lysing the cells in Laemmli sample buffer.

Hematopoietic stem cells (HSCs) are rare cells, with the mouse bone marrow containing only ~25,000 phenotypic long term repopulating HSCs. A Western ...

A11-positive &#946;-amyloid Oligomer Preparation and Assessment Using Dot Blotting Analysis

&#946;-amyloid (A&#946;) is a hydrophobic peptide with an intrinsic tendency to self-assemble into aggregates. Among various aggregates, A&#946; oligomer is widely accepted as the leading neurotoxin in the progress of Alzheimer&#39;s disease (AD) and is considered to be the crucial event in the pathogenesis of AD. Therefore, A&#946; oligomer inhibitors might prevent neurodegeneration and have the potential to be developed as disease-modifying treatments of AD. However, different formation protocols of A&#946; oligomer might lead to oligomers with different characteristics. Moreover, there are not many methods to effectively screen A&#946;1-42 oligomer inhibitors. An A11 antibody can react with a subset of toxic A&#946;1-42 oligomer with anti-parallel &#946;-sheet structures. In this protocol, we describe how to prepare an A11-positive A&#946;1-42 oligomer-rich sample from a synthetic A&#946;1-42 peptide in vitro and to evaluate relative amounts of A11-positive A&#946;1-42 oligomer in samples by a dot blotting analysis using A11 and A&#946;1-42-specific 6E10 antibodies. Using this protocol, inhibitors of A11-positive A&#946;1-42 oligomer can also be screened from semi-quantitative experimental results.

This protocol describes how to prepare A&#946; oligomers from a synthetic peptide in vitro and to evaluate relative amounts of A&#946; oligomer by a dot blotting analysis.

&#946;-amyloid (A&#946;) is a hydrophobic peptide with an intrinsic tendency to self-assemble into aggregates. Among various aggregates, A&#946; oligomer ...

Anaerobic Protein Purification and Kinetic Analysis via Oxygen Electrode for Studying DesB Dioxygenase Activity and Inhibition

Oxygen-sensitive proteins, including those enzymes which utilize oxygen as a substrate, can have reduced stability when purified using traditional aerobic purification methods. This manuscript illustrates the technical details involved in the anaerobic purification process, including the preparation of buffers and reagents, the methods for column chromatography in a glove box, and the desalting of the protein prior to kinetics. Also described are the methods for preparing and using an oxygen electrode to perform kinetic characterization of an oxygen-utilizing enzyme. These methods are illustrated using the dioxygenase enzyme DesB, a gallate dioxygenase from the bacterium Sphingobium sp. strain SYK-6.

Here we present a protocol for anaerobic protein purification, anaerobic protein concentration, and subsequent kinetic characterization using an oxygen electrode system. The method is illustrated using the enzyme DesB, a dioxygenase enzyme which is more stable and active when purified and stored in an anaerobic environment.

Oxygen-sensitive proteins, including those enzymes which utilize oxygen as a substrate, can have reduced stability when purified using traditional aerobic ...

Single Liposome Measurements for the Study of Proton-Pumping Membrane Enzymes Using Electrochemistry and Fluorescent Microscopy

Proton-pumping enzymes of electron transfer chains couple redox reactions to proton translocation across the membrane, creating a proton-motive force used for ATP production. The amphiphilic nature of membrane proteins requires particular attention to their handling, and reconstitution into the natural lipid environment is indispensable when studying membrane transport processes like proton translocation. Here, we detail a method that has been used for the investigation of the proton-pumping mechanism of membrane redox enzymes, taking cytochrome bo3 from Escherichia coli as an example. A combination of electrochemistry and fluorescence microscopy is used to control the redox state of the quinone pool and monitor pH changes in the lumen. Due to the spatial resolution of fluorescent microscopy, hundreds of liposomes can be measured simultaneously while the enzyme content can be scaled down to a single enzyme or transporter per liposome. The respective single enzyme analysis can reveal patterns in the enzyme functional dynamics that might be otherwise hidden by the behavior of the whole population. We include a description of a script for automated image analysis.

Here, we present a protocol to study the molecular mechanism of proton translocation across lipid membranes of single liposomes, using cytochrome bo3 as an example. Combining electrochemistry and fluorescence microscopy, pH changes in the lumen of single vesicles, containing single or multiple enzyme, can be detected and analyzed individually.

Proton-pumping enzymes of electron transfer chains couple redox reactions to proton translocation across the membrane, creating a proton-motive force used ...

Cortisol Measurement in Koala (Phascolarctos cinereus) Fur

Optimal methods of hormone extraction used to measure stress in animals across sample types are not always the same. Australia&#39;s iconic marsupial species, the koala (Phascolarctos cinereus), faces prolonged exposure to anthropogenic-induced stressors and assessment of chronic stress in wild populations is urgently warranted. One of the most effective ways to measure chronic stress is through analyzing the glucocorticoid hormone cortisol in hair or fur, as it supports physiological and behavioral responses. This laboratory validation study aims to test current techniques to validate an optimal hormone extraction method to be used as a non-invasive measure of cortisol in koala fur. It is recognized that using non-invasive techniques to measure stress hormones is preferred over traditional, invasive techniques due to their ideal practical and ethical standpoints. Additionally, it is comparatively easier to acquire fur from koalas than it is to acquire samples of their blood. This study used samples of koala fur acquired from the Adelaide Koala and Wildlife Hospital to run a number of hormone extraction techniques in an attempt to validate an optimal cortisol extraction method. Results showed that 100% methanol provided the most optimal solvent extraction compared to 100% ethanol or 100% isopropanol based on parallelism results. In conclusion, this method of cortisol extraction from koala fur provided a reliable non-invasive assay that could be used to study chronic stress in koalas.

Cortisol Measurement in Koala Phascolarctos cinereus Fur

We present a protocol to determine the optimal extraction solvent to measure cortisol from koala fur. The solvents used in this protocol are methanol, ethanol and isopropanol. Determining an optimal extraction solvent will aid in reliably measuring fur to determine the impact of chronic stress on koalas.

Optimal methods of hormone extraction used to measure stress in animals across sample types are not always the same. Australia&#39;s iconic marsupial ...

Robust Comparison of Protein Levels Across Tissues and Throughout Development Using Standardized Quantitative Western Blotting

Western blotting is a technique that is commonly used to detect and quantify protein expression. Over the years, this technique has led to many advances in both basic and clinical research. However, as with many similar experimental techniques, the outcome of Western blot analyses is easily influenced by choices made in the design and execution of the experiment. Specific housekeeping proteins have traditionally been used to normalize protein levels for quantification, however, these have a number of limitations and have therefore been increasingly criticized over the past few years. Here, we describe a detailed protocol that we have developed to allow us to undertake complex comparisons of protein expression variation across different tissues, mouse models (including disease models), and developmental timepoints. By using a fluorescent total protein stain and introducing the use of an internal loading standard, it is possible to overcome existing limitations in the number of samples that can be compared within experiments and systematically compare protein levels across a range of experimental conditions. This approach expands the use of traditional western blot techniques, thereby allowing researchers to better explore protein expression across different tissues and samples.

This method describes a robust and reproducible approach for the comparison of protein levels in different tissues and at different developmental timepoints using a standardized quantitative western blotting approach.

Western blotting is a technique that is commonly used to detect and quantify protein expression. Over the years, this technique has led to many advances ...

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Robust Comparison of Protein Levels Across Tissues and Throughout Development Using Standardized Quantitative Western Blotting