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

Summary

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.

Abstract

The IRF3 transcription factor is critical for the first line of defense against pathogens mainly through interferon β 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.

Introduction

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 IFNβ, 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 IFIT1/2/3^1-3. 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....

Protocol

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

1. Maintain A549 cells in culture in a 15 cm plate at 37 °C/5% CO₂ in 20 ml F12K/Ham medium containing 10% heat-inactivated fetal bovine serum (HI-FBS) and 1% L-glutamine (complete F12K/Ham medium).
   NOTE: All the solutions used for cell culture and treatments must be sterile.
2. At 24 ....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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