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).

MERK: Protokollen er beskrevet her bruker A549 celler infisert med Sendai virus (SeV). Imidlertid fungerer også protokollen for SDS-PAGE og nativ PAGE med alle humane og murine celletyper som ble testet hittil, særlig myeloide celler stimulert med forskjellige IRF3-aktiverende stimuli 9,15,19,24,25. 1. Infeksjon av A549-celler <ol><li> Oppretthold A549-celler i kultur i en 15 cm plate ved 37 ° C / 5% CO2 i 20 ml F12K / Ham medium inneholdende 10% varme-inaktivert f...

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	<li>Juang, Y. 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=Primary+activation+of+interferon+A+and+interferon+B+gene+transcription+by+interferon+regulatory+factory-3.">Primary activation of interferon A and interferon B gene transcription by interferon regulatory factory-3.</a> Proc Natl Acad Sci U S A. 95 (17), 9837-9842 (1998).</li><li>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=A+role+for+casein+kinase+II+phosphorylation+in+the+regulation+of+IRF-1+transcriptional+activity.">A role for casein kinase II phosphorylation in the regulation of IRF-1 transcriptional activity.</a> Mol Cell Biochem. 191 (1-2), 169-180 (1999).</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=Transcriptional+profiling+of+interferon+regulatory+factor+3+target+genes:+direct+involvement+in+the+regulation+of+interferon-stimulated+genes.">Transcriptional profiling of interferon regulatory factor 3 target genes: direct involvement in the regulation of interferon-stimulated genes.</a> J Virol. 76 (11), 5532-5539 (2002).</li><li>Thompson, M. R., Kaminski, J. J., Kurt-Jones, E. A., Fitzgerald, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pattern+recognition+receptors+and+the+innate+immune+response+to+viral+infection.">Pattern recognition receptors and the innate immune response to viral infection.</a> Viruses. 3 (6), 920-940 (2011).</li><li>Grandvaux, N., tenOever, B. R., Servant, M. J., 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=The+interferon+antiviral+response:+from+viral+invasion+to+evasion.">The interferon antiviral response: from viral invasion to evasion.</a> Curr Opin Infect Dis. 15 (3), 259-267 (2002).</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+Distinct+Signaling+Pathways+Leading+to+the+Phosphorylation+of+Interferon+Regulatory+Factor+3.">Identification of Distinct Signaling Pathways Leading to the Phosphorylation of Interferon Regulatory Factor 3.</a> J Biol Chem. 276 (1), 355-363 (2001).</li><li>Qin, B. 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=Crystal+structure+of+IRF-3+reveals+mechanism+of+autoinhibition+and+virus-induced+phosphoactivation.">Crystal structure of IRF-3 reveals mechanism of autoinhibition and virus-induced phosphoactivation.</a> Nat Struct Biol. 10 (11), 913-921 (2003).</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=X-ray+crystal+structure+of+IRF-3+and+its+functional+implications.">X-ray crystal structure of IRF-3 and its functional implications.</a> Nat Struct Biol. 10 (11), 922-927 (2003).</li><li>Mori, 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=Identification+of+Ser-386+of+interferon+regulatory+factor+3+as+critical+target+for+inducible+phosphorylation+that+determines+activation.">Identification of Ser-386 of interferon regulatory factor 3 as critical target for inducible phosphorylation that determines activation.</a> J Biol Chem. 279 (11), 9698-9702 (2004).</li><li>Clement, J. 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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Anal Biochem. 72, 248-254 (1976).</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 Cells. 6 (4), 375-388 (2001).</li><li>tenOever, B. R., Servant, M. 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>

Protokollen Vi beskriver her består av en kombinasjon av høy oppløsning og SDS-PAGE og native-PAGE koblet til bruken av flere phosphospecific antistoffer for å skille monomere / dimere og phosphoforms I-IV av IRF3. Hensiktsmessig påvisning av disse IRF3 artene er nødvendig for å fullstendig karakterisering IRF3 aktivering i en bestemt innstilling. For eksempel, LPS stimulering av aktiverte makrofager fører til dannelse av dimer, Ser396 / 398 fosforylert IRF3 som oppviser en hypofosforylerte (skjema II), men ikke...

<html> Den overalt og konstitutivt uttrykt transkripsjonsfaktor Interferon (IFN) regulerende faktor 3 (IRF3) er kritisk for den første linjen i forsvaret mot patogener hovedsakelig gjennom induksjon av IFNβ, men også gjennom induksjon av chemokine (CC motiv) ligand 5 (CCL5 ) og flere antivirale proteiner inkludert IFN-indusert protein med tetratricopeptide gjentar IFIT1 / 2/3 1-3. IRF3 aktivering er rapportert etter infeksjon med en rekke virus, eller eksponering for polyinosinic-polycytidylic syre (poly I: C) eller lipopolysakkarid (LPS) 4. Viktigere, har mest studerte virus utviklet mekanismer for å unngå den IRF3-mediert respons, og dermed unnslippe verten medfødte immunforsvaret 5. Således, overvåking IRF3 aktivering er av stor betydning for å forstå de molekylære mekanismer av medfødte antivirale vertsforsvar, men også for å identifisere den strategi som brukes av virus for å motvirke denne reaksjon. ent &quot;&gt; Mange publiserte rapporter gir imidlertid bare en begrenset analyse av IRF3 aktivering utført ved overvåkning av IRF3-target geninduksjon (IFNB1 og IFIT1) og / eller luciferase reporter-genet koblet til analysen lav oppløsning natriumdodecylsulfat polyakrylamid gelelektroforese (SDS- PAGE) analyse av IRF3. Men mange biokjemiske studier, analyse av virkemåten til forskjellige IRF3 mutanter og belysning av IRF3 krystallstrukturen 6-11 har bidratt til å etablere at IRF3 utsettes for et komplekst sett av sekvensielle post-translasjonelle modifikasjoner av fosforylering ved flere steder. Settet av fosforylering involvert i IRF3 aktivering ser ut til å være avhengig av stimulus og mest sannsynlig på celletype. I uinfiserte celler, IRF3 eksisterer som ikke-fosforylerte og hypofosforylerte arter som inneholder phosphoresidues, inkludert Thr135 og Ser173, i ett -198 aa N-terminal region 6,12-14. Opphopning av denne hypofosforylerte form av IRF3 er indusert av stress-induse, vekstfaktorer og DNA-skadende midler 6. Fosforylering av Ser / Thr-rester på den C-terminale region av IRF3 inneholdende trans domenet utløses etter aktivering av virus, poly I: C eller LPS i en celletype-avhengig måte 15-17. C-terminal fosforylering av IRF3 innebærer ikke mindre enn 7 forskjellige phosphoacceptor nettsider organisert i to hoved klynger, Ser385 / Ser386 og Ser396 / Ser398 / Ser402 / Thr404 / Ser405, som hver bidrar til IRF3 aktivisering gjennom dimerization, kjernekraft akkumulering, tilknytning til CREB -binding protein (CBP) / P300 coactivators, DNA binding til IFN sensitiv respons element (SBR) konsensussekvenser og trans av målgener 9,10,17-19. Fosforylering av Thr390 er også tenkt å bidra til virus-indusert IRF3 aktivering 20. Massespektrometri analyser av IRF3 har vist at Ser386, Thr390, Ser396 og Ser402 rester er direkte phosphorylated ved hemmer av kB kinase ε (IKKε) / TANK-bindende kinase 1 (TBK1) kinaser 9,10. Fosforylering ved den C-terminale rester er også nødvendig for terminering av IRF3 aktivering polyubiquitination og proteasom-mediert nedbrytning 10. Denne prosessen er også avhengig av fosforylering ved Ser339, noe som er nødvendig for rekruttering av propyl-isomerase Pin1 10,11. IRF3 arter inneholder minst fosfor-Ser339 / 386/396 rester anses hyperfosforylert former. Den nøyaktige sekvensen og funksjonen til hvert område er fortsatt et spørsmål om diskusjon 10,21. Det er nå klart at aktivert IRF3 representerer ikke en homogen stat, men at ulike aktivert arter utviser tydelige fosforyleringsseter eller dimerisasjon egenskaper eksisterer 10,22. For å gi en fullstendig forståelse av IRF3 aktivering som svar på spesifikke patogener, er det derfor nødvendig å characterize hvilke av de aktiverte arter er indusert. Induksjon av IRF3 målgener, IFNB1 og IFIT1, har vist seg å gi en pålitelig avlesing for IRF3 aktivering. Men overvåking uttrykk av disse genene skiller ikke mellom ulike aktiverings tilstander av IRF3. En omfattende analyse av IRF3 aktiverings stater i en bestemt innstilling bygger på detaljert karakterisering av sin fosforylering og dimerization status 10. Ufosforylerte (skjema I), hypofosforylerte (skjema II) og hyperfosforylert (skjemaer III og IV) IRF3 former 6,18,23 kan løses ved redusert mobilitet i høy oppløsning SDS-PAGE analyse. Monomere og dimeriske IRF3 arter kan effektivt identifisert av innfødte-PAGE analyse. Disse metodene er kraftig forbedret når de brukes i kombinasjon med phosphospecific antistoffer rettet mot forskjellige IRF3 phosphoacceptor nettsteder. Standard protokoller tillate en dårlig oppløsning påproteiner som ikke tillater effektiv separasjon av distinkte IRF3 fosforylerte formene. Her beskriver vi i detalj en fremgangsmåte for å oppnå den høyeste oppløsningen for å overvåke induksjon av forskjellige virus-aktivert IRF3 arter ved hjelp av SDS-PAGE koblet med nativ-PAGE i kombinasjon med immunblot ved hjelp av total og phosphospecific antistoffer. In vivo diskriminering mellom de forskjellige aktiverte former IRF3 utføres basert på deres mobilitet skift observert på SDS-PAGE. I tillegg kan IRF3 monomer og dimer være preget av ikke-denaturerende elektroforese. Kombinasjonen av disse to komplementære teknikker med immunoblot viser seg å være en rimelig og sensitiv tilnærming til å skaffe all nødvendig informasjon for en fullstendig analyse av fosforylering-mediert aktivering av IRF3.

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		</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.

Figur 2 viser en typisk immunoblot bilde av IRF3 oppdaget med IRF3 totale antistoffer og IRF3-phosphospecific antistoffer mot Ser396 og Ser398 etter oppløsningen av WCE av høyoppløselig SDS-PAGE. I ikke-stimulerte A549-celler, er IRF3 detektert som to bånd ved 50 og 53 kDa på SDS-PAGE svarende til den ikke-fosforylert (form I) og den hypofosforylerte (skjema II) arter av IRF3. Eksponering av A549 celler til SEV for 3 - 9 t resulterer i en tidsavhengig forskyvning ti...

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.

En High Resolution metode for å overvåke Fosforylering avhengig Aktivering av IRF3

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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