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. ...

The overall goal of this procedure is to allow a detailed analysis of the phosphorylation-dependent activation of the IRF3 transcription factor. So this method can help answer key questions regarding the interferon mediate innate immune response. Such as a pathogen activates or invades the IRF3 dependent interferon beta expression.The main advantage of this technique is that is constitutes affordable and sensitive approach to distinguish the different IRF3 activated form that may arise following different stimulations. Though this method provides insight into virus infection of human cells it can also be applied to mouse or human tissues subjected to other pathogens or pathogen-associated molecular patterns. Demonstrating the procedure will be Alexa Robitaille and Melissa Mariani, two cell Simulink experts from my laboratory.The A549 cells used in this procedure are maintained in culture in 15 centimeter plate at 37 degrees Celsius and 5%carbon dioxide in complete F-12K Ham medium. Twenty four hours before infection, trypsinize the cells as described in the protocol text and transfer the cells to a 15 milliliter conical tube. Centrifuge at 350 x G for three minutes at room temperature.Remove the supernatant and re-suspend the cell pellet in eight milliliters of complete F-12K Ham medium to obtain a homogeneous single-cell suspension. After counting the cells using a hemoctyometer, seed the cells at a density of one times tenth to the sixth cells per 60 millimeter plate, and four milliliters of preheated complete F-12K Ham medium. Incubate for 20 to 24 hours at 37 degrees Celsius, five percent carbon dioxide.After 20 to 24 hours the cells should form a 90%confluent monolayer. Remove the medium and wash the cells with two milliliters of serum-free F-12K Ham medium, or SFM. Add two milliliters of fresh SFM per 60 millimeter plate.Dilute to thawed aliquot of Sendai virus in preheated SFM to obtain 60 hemagglutination units, or HAU, per 100 microliters. Mix by pipetting up and down gently, and then add 100 microliters of diluted virus drop by drop to each plate to perform the infection at 40 HAU per 10 to the sixth cells. Do not add virus in the non-infected control plate.Incubate the cells in the incubator at 37 degrees Celsius, five percent carbon dioxide. During the first hour of infection, agitate the plates by hand three or four times. At two hours post-infection add two milliliters of F-12K Ham medium containing 20%heat and activated FDS, to obtain a final concentration of 10%heat and activated FDS.Incubate the cells in the incubator for one, four, and seven more hours to reach total infection times of three, six, and nine hours respectively. Cells will be harvested at each of these time points for preparation of whole cell extracts as demonstrated in the next video segment. Begin this procedure by removing the infection medium.Harvest the cells by scraping in one milliliter of ice-cold DPBS. Transfer the cell suspension to a pre-chilled 1.5 milliliter centrifuge tube. Pellet the cells by centrifugation at 16, 000 x g at 4 degrees Celsius for 20 seconds.And carefully decant all traces of DPBS. Re-suspend the cell pellet in 70 microliters of lysis buffer. Incubate on ice for 20 minutes.Flash-freeze the lysate by incubation in a liquid nitrogen bath for 15 seconds. Then thaw the lysate at room temperature until it is completely melted. Vortex for ten seconds.Repeat this freeze-thaw-vortex cycle three times. Centrifuge at 16, 000 x g at four degrees Celsius for 20 minutes. Transfer the supernatant, which is the whole cell extract, to a new pre-chilled 1.5 milliliter centrifuge tube.It is important to keep the whole cell extracts on ice at all times. To resolve the whole cell extracts by high-resolution SDS-PAGE, prepare three denaturing electrophoresis gels, two 16 centimeter gels for the detection of IRF3 forms, and one 8.5 centimeter gel for the detection of Sendai virus proteins. Load 14 microliters of molecular weight standard in one well of each 16 centimeter gel.Next load 30 micrograms of denatured whole cell extract per well of the two 16 centimeter gels. Load seven microliters of molecular weight standard in one well of the 8.5 centimeter gel. Load eight to ten micrograms of denatured whole cell extract per well of the 8.5 centimeter gel.Run the gels at 30 milliamps constant current until the migration front reaches the bottom of the gel. Migration technically lasts approximately three hours for a 16 centimeter gel, and 45 minutes for an 8.5 centimeter gel. Begin this analysis by preparing a non-denaturing resolving gel of a minimum of 8.5 centimeters in length, following the procedure in the protocol text.Press the running apparatus into ice approximately to the level of the lower chamber electrophoresis buffer, but make sure the gel is not in the ice. Pre-run the gel at 40 milliamps constant current for 30 minutes on ice. During the pre-run, mix the whole cell extracts kept on ice one-to-one with Two-X Native-PAGE loading buffer.Immediately at the end of the pre-run, load eight to ten micrograms of whole cell extract on the gel. It is important to load the samples close to the bottom of the well so that the banding is not disturbed. Run the gel at 25 milliamps constant current on ice as demonstrated for the pre-run until the migration front reaches the bottom of the gel.This will take approximately 40 minutes. At the completion of all gel runs, the proteins are transferred to nitro-cellulose membranes and fixed. The transfer procedure is described in the accompanying protocol text.The three SDS-PAGE membranes, but not the Native-PAGE membrane, are stained with red Ponceau solution to identify the molecular weight markers. After incubating all four membranes from SDS-PAGE and Native-PAGE in blocking solution, incubate the membranes with the primary antibodies according to the sequential order shown in this diagram. Membranes must under go five five-minute washes in PBS-T before incubation with the appropriate horseradish peroxidase conjugated secondary antibodies.After removing traces of tween with PBS, incubate the membranes for one minute in the volume of enhanced chemiluminescence reagent sufficient to fully cover the membranes. Use filter paper to dry the membranes and then place them in the luminescent image analyzer to visualize the immuno-reactive bands. Before incubation with the next primary antibodies, wash the membranes three times in PBS for five minutes each time.When stripping is required between antibodies, incubate the membranes in pre-warmed stripping solution at 50 degrees Celsius for 20 minutes under regular agitation. Details of the immunoblot analysis can be found in the accompanying protocol text. Shown here is a typical immunoblot image of IRF3 detected with IRF3 total antibodies and IRF3 phosphospecific antibodies against Ser396 and Ser398, after high-resolution of whole cell extracts.In unstimulated cells, IRF3 is detected as two bands corresponding to the nonphosphorylated, and the hypophosphorylated species of IRF3. Exposure of cells to Sendai virus results in a time-dependent shift to slowly migrating hyperphosphorylated forms three and four, which are also specifically immunodetected using the phosphospecific antibodies against Ser396 and Ser398. This next figure shows a profile of IRF3 detection obtained from whole cell extracts resolved by native PAGE, followed by immunoblot using anti-IRF3 NES antibodies, and phosphospecific antibodies against Ser386.In unstimulated cells IRF3 is detected as a single band corresponding to the momomeric form. Upon infection with Sendai virus the levels of IRF3 monomer decrease with a concomitant accumulation of a slowly migrating band that corresponds to the dimeric form of IRF3, which is also specifically immunodetected using phosphospecific antibodies against Ser386. While attempting this procedure, it's important to remember that the composition and length of the different gels is critical to achieve an appropriate resolution of the IRF3 phosphoforms.Following this procedure, other methods, like DNA binding or gene reporter acids can be performed in order to assess IRF3 activity. After watching this video you should have a good understanding of how to distinguish the different active forms of IRF3. Don't forget that working with pathogens may be hazardous and manipulations should be performed in accordance to the biosafety guidelines of your host institution.

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