Name: a high resolution method to monitor phosphorylation-dependent activation of irf3
Uploaded: 2016-01-24T15:00:00
Description: Watch this Scientific Journal Video about A High Resolution Method to Monitor Phosphorylation-dependent Activation of IRF3 at JoVE.com

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

NOTE: The protocol is described here using A549 cells infected with Sendai virus (SeV). However, the protocol for SDS-PAGE and native PAGE also works with all human and murine cell types tested so far, particularly myeloid cells stimulated with various IRF3-activating stimuli 9,15,19,24,25.
1. Infection of A549 Cells
<ol>
	<li>Maintain A549 cells in culture in a 15 cm plate at 37 &#176;C/5% CO2 in 20 ml F12K/Ham medium containing 10% heat-inactivated fetal bovine serum ...

<ol>
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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=Phosphorylation+of+IRF-3+on+Ser+339+generates+a+hyperactive+form+of+IRF-3+through+regulation+of+dimerization+and+CBP+association.">Phosphorylation of IRF-3 on Ser 339 generates a hyperactive form of IRF-3 through regulation of dimerization and CBP association.</a> J Virol. 82 (8), 3984-3996 (2008).</li><li>Saitoh, 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=Negative+regulation+of+interferon-regulatory+factor+3-dependent+innate+antiviral+response+by+the+prolyl+isomerase+Pin1.">Negative regulation of interferon-regulatory factor 3-dependent innate antiviral response by the prolyl isomerase Pin1.</a> Nat Immunol. 7 (6), 598-605 (2006).</li><li>Wathelet, M. 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=Virus+infection+induces+the+assembly+of+coordinately+activated+transcription+factors+on+the+IFN-beta+enhancer+in+vivo.">Virus infection induces the assembly of coordinately activated transcription factors on the IFN-beta enhancer in vivo.</a> Mol Cell. 1 (4), 507-518 (1998).</li><li>Karpova, A. Y., Trost, M., Murray, J. M., Cantley, L. C., Howley, P. M. <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-3+is+an+in+vivo+target+of+DNA-PK.">Interferon regulatory factor-3 is an in vivo target of DNA-PK.</a> Proc Natl Acad Sci U S A. 99 (5), 2818-2823 (2002).</li><li>Zhang, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+TAK1-JNK+cascade+is+required+for+IRF3+function+in+the+innate+immune+response.">The TAK1-JNK cascade is required for IRF3 function in the innate immune response.</a> Cell Res. 19 (4), 412-428 (2009).</li><li>Solis, 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=Involvement+of+TBK1+and+IKKepsilon+in+lipopolysaccharide-induced+activation+of+the+interferon+response+in+primary+human+macrophages.">Involvement of TBK1 and IKKepsilon in lipopolysaccharide-induced activation of the interferon response in primary human macrophages.</a> Eur J Immunol. 37 (2), 528-539 (2007).</li><li>Soucy-Faulkner, 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=Requirement+of+NOX2+and+reactive+oxygen+species+for+efficient+RIG-I-mediated+antiviral+response+through+regulation+of+MAVS+expression.">Requirement of NOX2 and reactive oxygen species for efficient RIG-I-mediated antiviral response through regulation of MAVS expression.</a> PLoS Pathog. 6 (6), e1000930 (2010).</li><li>Lin, R., Heylbroeck, C., Pitha, P. M., Hiscott, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus-dependent+phosphorylation+of+the+IRF-3+transcription+factor+regulates+nuclear+translocation,+transactivation+potential,+and+proteasome-mediated+degradation.">Virus-dependent phosphorylation of the IRF-3 transcription factor regulates nuclear translocation, transactivation potential, and proteasome-mediated degradation.</a> Mol Cell Biol. 18 (5), 2986-2996 (1998).</li><li>Yoneyama, 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=Direct+triggering+of+the+type+I+interferon+system+by+virus+infection:+activation+of+a+transcription+factor+complex+containing+IRF-3+and+CBP/p300.">Direct triggering of the type I interferon system by virus infection: activation of a transcription factor complex containing IRF-3 and CBP/p300.</a> EMBO J. 17 (4), 1087-1095 (1998).</li><li>Servant, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+the+minimal+phosphoacceptor+site+required+for+in+vivo+activation+of+interferon+regulatory+factor+3+in+response+to+virus+and+double-stranded+RNA.">Identification of the minimal phosphoacceptor site required for in vivo activation of interferon regulatory factor 3 in response to virus and double-stranded RNA.</a> J Biol Chem. 278 (11), 9441-9447 (2003).</li><li>Bergstroem, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+novel+in+vivo+virus-targeted+phosphorylation+site+in+interferon+regulatory+factor-3+(IRF3).">Identification of a novel in vivo virus-targeted phosphorylation site in interferon regulatory factor-3 (IRF3).</a> J Biol Chem. 285 (32), 24904-24914 (2010).</li><li>Takahasi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ser386+phosphorylation+of+transcription+factor+IRF-3+induces+dimerization+and+association+with+CBP/p300+without+overall+conformational+change.">Ser386 phosphorylation of transcription factor IRF-3 induces dimerization and association with CBP/p300 without overall conformational change.</a> Genes Cells. 15 (8), 901-910 (2010).</li><li>Noyce, R. S., Collins, S. E., Mossman, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+modification+of+interferon+regulatory+factor+3+following+virus+particle+entry.">Differential modification of interferon regulatory factor 3 following virus particle entry.</a> J Virol. 83 (9), 4013-4022 (2009).</li><li>McCoy, C. E., Carpenter, S., Palsson-McDermott, E. M., Gearing, L. J., O'Neill, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucocorticoids+inhibit+IRF3+phosphorylation+in+response+to+Toll-like+receptor-3+and+-4+by+targeting+TBK1+activation.">Glucocorticoids inhibit IRF3 phosphorylation in response to Toll-like receptor-3 and -4 by targeting TBK1 activation.</a> J Biol Chem. 283 (21), 14277-14285 (2008).</li><li>Oliere, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HTLV-1+evades+type+I+interferon+antiviral+signaling+by+inducing+the+suppressor+of+cytokine+signaling+1+(SOCS1).">HTLV-1 evades type I interferon antiviral signaling by inducing the suppressor of cytokine signaling 1 (SOCS1).</a> PLoS Pathog. 6 (11), e1001177 (2010).</li><li>Kato, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+type-specific+involvement+of+RIG-I+in+antiviral+response.">Cell type-specific involvement of RIG-I in antiviral response.</a> Immunity. 23 (1), 19-28 (2005).</li><li>Bradford, M. 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J., Grandvaux, N., Lin, R., Hiscott, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognition+of+the+measles+virus+nucleocapsid+as+a+mechanism+of+IRF-3+activation.">Recognition of the measles virus nucleocapsid as a mechanism of IRF-3 activation.</a> J Virol. 76 (8), 3659-3669 (2002).</li><li>Bibeau-Poirier, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+the+I{kappa}B+Kinase+(IKK)-Related+Kinases+Tank-Binding+Kinase+1/IKKi+and+Cullin-Based+Ubiquitin+Ligases+in+IFN+Regulatory+Factor-3+Degradation.">Involvement of the I{kappa}B Kinase (IKK)-Related Kinases Tank-Binding Kinase 1/IKKi and Cullin-Based Ubiquitin Ligases in IFN Regulatory Factor-3 Degradation.</a> J Immunol. 177 (8), 5059-5067 (2006).</li><li>Grandvaux, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustained+Activation+of+Interferon+Regulatory+Factor+3+during+Infection+by+Paramyxoviruses+Requires+MDA5.">Sustained Activation of Interferon Regulatory Factor 3 during Infection by Paramyxoviruses Requires MDA5.</a> J Innate Immun. 6 (5), 650-662 (2014).</li></ol>

The protocol we describe here consists of a combination of high-resolution SDS-PAGE and native-PAGE coupled to the use of several phosphospecific antibodies to distinguish the monomeric/dimeric and phosphoforms I-IV of IRF3. Appropriate detection of these IRF3 species is essential to fully characterize IRF3 activation in a specific setting. For instance, LPS stimulation of activated macrophages leads to the formation of dimeric, Ser396/398 phosphorylated IRF3 that exhibits a hypophosphorylated (form II), but not hyperpho...

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

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

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

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

Figure 2 shows a typical immunoblot image of IRF3 detected with IRF3 total antibodies and IRF3-phosphospecific antibodies against Ser396 and Ser398 after resolution of WCE by high-resolution SDS-PAGE. In unstimulated A549 cells, IRF3 is detected as two bands at 50 and 53 kDa on the SDS-PAGE corresponding to the non-phosphorylated (form I) and the hypophosphorylated (form II) species of IRF3. Exposure of A549 cells to SeV for 3 - 9 hr results in a time-dependent shift to s...

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

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

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

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

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