Name: native polyacrylamide gel electrophoresis immunoblot analysis of endogenous irf5 dimerization
Uploaded: 2019-10-06T15:00:00
Description: Watch this Scientific Journal Video about Native Polyacrylamide Gel Electrophoresis Immunoblot Analysis of Endogenous IRF5 Dimerization at JoVE.com

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
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	<li>Maintain CAL-1 cell cultures in a T75 flask at 37 &#176;...

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+and+challenges+in+targeting+IRF5,+a+key+regulator+of+inflammation.">Advances and challenges in targeting IRF5, a key regulator of inflammation.</a> FEBS Journal. 286 (9), 1624-1637 (2019).</li><li>Chow, K. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+and+Overlapping+Immune+Programs+Regulated+by+IRF3+and+IRF5+in+Plasmacytoid+Dendritic+Cells.">Differential and Overlapping Immune Programs Regulated by IRF3 and IRF5 in Plasmacytoid Dendritic Cells.</a> The Journal of Immunology. 201 (10), 3036-3050 (2018).</li><li>Cheng, T. 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=Differential+Activation+of+IFN+Regulatory+Factor+(IRF)-3+and+IRF-5+Transcription+Factors+during+Viral+Infection.">Differential Activation of IFN Regulatory Factor (IRF)-3 and IRF-5 Transcription Factors during Viral Infection.</a> The Journal of Immunology. 176 (12), 7462-7470 (2006).</li><li>Chang Foreman, H. C., Van Scoy, S., Cheng, T. F., Reich, N. 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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=Interferon+regulatory+factor+5+activation+in+monocytes+of+systemic+lupus+erythematosus+patients+is+triggered+by+circulating+autoantigens+independent+of+type+I+interferons.">Interferon regulatory factor 5 activation in monocytes of systemic lupus erythematosus patients is triggered by circulating autoantigens independent of type I interferons.</a> Arthritis and Rheumatology. 64 (3), 788-798 (2012).</li><li>De, 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=B+Cell-Intrinsic+Role+for+IRF5+in+TLR9/BCR-Induced+Human+B+Cell+Activation,+Proliferation,+and+Plasmablast+Differentiation.">B Cell-Intrinsic Role for IRF5 in TLR9/BCR-Induced Human B Cell Activation, Proliferation, and Plasmablast Differentiation.</a> Frontiers in Immunology. 8, 1938 (2017).</li><li>Fabie, 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=IRF-5+Promotes+Cell+Death+in+CD4+T+Cells+during+Chronic+Infection.">IRF-5 Promotes Cell Death in CD4 T Cells during Chronic Infection.</a> Cell Reports. 24 (5), 1163-1175 (2018).</li><li>Cushing, 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=IRAK4+kinase+activity+controls+Toll-like+receptor-induced+inflammation+through+the+transcription+factor+IRF5+in+primary+human+monocytes.">IRAK4 kinase activity controls Toll-like receptor-induced inflammation through the transcription factor IRF5 in primary human monocytes.</a> Journal of Biological Chemistry. 292 (45), 18689-18698 (2017).</li><li>Li, C., Arakawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+native+polyacrylamide+gel+electrophoresis+for+protein+analysis:+Bovine+serum+albumin+as+a+model+protein.">Application of native polyacrylamide gel electrophoresis for protein analysis: Bovine serum albumin as a model protein.</a> International Journal of Biological Macromolecules. 125, 566-571 (2019).</li><li>Iwamura, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+IRF-3/-7+kinase+and+NF-kappaB+in+response+to+double-stranded+RNA+and+virus+infection:+common+and+unique+pathways.">Induction of IRF-3/-7 kinase and NF-kappaB in response to double-stranded RNA and virus infection: common and unique pathways.</a> Genes to Cells. 6 (4), 375-388 (2001).</li><li>Subhadarshanee, B., Mohanty, A., Jagdev, M. 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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.

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			<td>21985023</td>
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			<td>Anti-IRF5 antibody</td>
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			<td>EDTA Buffer, pH 8, 0.5 M 4 X 100 mL</td>
			<td>Life Technologies</td>
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			<td>Life Technologies</td>
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			<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 &#8805;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 ...

This protocol utilizes a widely-used technique along with reagents and tools that can be acquired commercially to assess endogenous IRF5 activation during the early events of stimulation. It can easily be applied for probing IRF5 biology in other cellular context. While native PAGE is a widely-used technique, obtaining clear, robust, interpretable, and reproducible results can be technically challenging.Using a commercial running buffer and gel system helps to minimize variability. And paying attention to details is essential, and experience is key to success in obtaining good results. This modified native PAGE protocol involves many steps with subtle details that are key to success.Visual demonstration can show these details that would be beneficial for researchers. Maintain CAL-1 cell culture in a T75 flask at 37 degrees Celsius and 5%carbon dioxide, with complete RPMI 1640 medium. When ready to stimulate the cells, transfer them to a 50-milliliter conical tube and centrifuge them at 200 x g for five minutes.Remove the supernatant, and re-suspend the pellet in medium to obtain a homogeneous single cell suspension. Then account the cells with a hemocytometer, and seed them at a density of 1, 000, 000 cells per well in a six-well plate with four milliliters of pre-heated medium in each well. Incubate the cells for 20 to 24 hours to allow the confluency to reach 90 to 95%The next day, stimulate the cells by adding four microliters of one milligram per milliliter R848 per well, making sure to leave a control well with cells and no R848 treatment.Gently rock the plate from side to side to evenly disperse the R848, then incubate the cells for 2 to 16 hours at 37 degrees Celsius and 5%carbon dioxide. After the incubation, transfer the cell suspensions from the plate into 5-milliliter centrifuge tubes. Centrifuge the cells at 200 x g for five minutes, then remove the supernatant, and re-suspend the pellet in one milliliter of PBS.Transfer the cell suspension into a 1.5-milliliter tube, and spin it down at 12, 000 x g for 30 to 60 seconds at 4 degrees Celsius, then carefully remove the supernatant. Prepare the lysis buffer according to the manuscript directions, and keep it on ice until ready to use. Re-suspend the cell pellet in 30-microliters of ice-cold lysis buffer, and pipette up and down to mix.Then incubate the tube on ice for 15 to 20 minutes. Clarify the lysate by centrifuging at 12, 000 x g for 15 to 20 minutes at 4 degrees Celsius, and transfer the supernatant to a pre-chilled 1.5-milliliter tube, making sure to keep the extracts on ice at all times. Then, measure the protein concentration using Bradford Reagent.Prepare the upper-and lower-chamber electrophoresis buffers according to the manuscript directions, and rinse a 3-12%native PAGE gel thoroughly with water, without distorting the wells Extra care should be taken when handling the sodium deoxycholate. PPE for protection such as lab coat, safety goggles, and mask are required for handling the chemical. Then, set the gel into the Mini Gel Tank, remove the comb, add the prepared upper-and lower-chamber electrophoresis buffers, and pre-run it in a 4 degrees Celsius cold room or on ice at 150 volts for 30 minutes.Meanwhile, prepare the samples for loading by mixing the cellular proteins on ice with 4X Native Sample Buffer. Once the pre-run is complete, load 10 to 15 micrograms of protein to each well, and run the gel for 30 minutes at 85 volts, followed by two hours at 150 volts. Then, soak the gel in SDS running buffer for 30 minutes at room temperature.Activate the polyvinylidene difluoride membrane by soaking it in methanol for approximately five minutes. Make a cut on one corner of the membrane to indicate its orientation, and assemble the transfer sandwich according to the manufacturer's protocol. Place the transfer cassette into the tank and transfer at 20 volts for one hour on ice.Then remove the membrane from the cassette with plastic forceps and block the membrane in blocking buffer at room temperature for 45 minutes on a rocking shaker. Next, incubate the membrane with the primary antibody at 4 degrees Celsius overnight or at room temperature for two hours and wash it with 1XTBST washing buffer for three minutes while rocking. Then incubate the membrane with the secondary antibody again using the 1XTBST washing buffer at room temperature for 45 minutes, repeating the washes.Then scan the blot using an appropriate gel documentation system. Using this protocol, CAL-1 cells that were either stimulated or unstimulated with R848 were analyzed with an immunoblot. In unstimulated CAL-1 cells, IRF5 was detected as a single band on the native PAGE corresponding to its monomeric form.For the stimulated cells, the level of IRF5 monomer decreased, while the level of the dimer increased. An immunoblot with anti-IRF5 antibody was performed on IRF5 over-expressing 293 T cells transfected with various constructs. No IRF5 was detected in the untransfected control, demonstrating the specificity of the anti-IRF5 antibody.A single band corresponding to monomeric IRF5 was only detected in the 293 T cells over-expressing IRF5. When constructs encoding IRF5 activating proteins were co-transfected, a slowly-migrating band corresponding to the dimeric form of IRF5 appeared. However, NMDA5, a related protein to RIG-I, did not induce IRF5 dimerization.The use of Bis-Tris gradient gels is crucial, likely due to the specific pH and chemical composition of this gel electrophoretic systems that allowed separation of monomeric and dimeric forms of IRF5. Also, lysing and preserving cell lysates in non-denaturing native sample buffer retains native protein structures. Here we used a commercially-available one that is tailored for native PAGE acids.An additional method that compliments greatly with this is, is to utilize the ImageStream imaging flow cytometry system to assess IRF5 nuclear translocation, which is the substance that in IRF5 activation after dimerization. When combined with our protocol, it serves as an other kind of test to validate the steps involved in IRF5 activation. Being a key regulator of the inflammatory response, IRF5 plays important role in infection and immunity, autoimmune diseases, cancer, and many other diseases important for human health.There are also efforts in developing therapeutics to target IRF5 and related transcription factors. This protocol will allow researchers in diverse fields to understand and probe IRF5 biology.

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