Anti phospho irf3 antibody

Cellular extracts or eluates from immunoprecipitations were diluted in 2x Laemmli sample buffer (BioRad), separated by SDS-PAGE in 10% Tris-glycine-gels and blotted onto a PVDF-membrane (pore size 0.2 µM, BioRad). After blocking in 5% milk in TBS with 0.1%Tween 20 or 5% BSA (when anti-phospho-IRF3 antibody was used consecutively) for 1 hour at RT. Membranes were then incubated over night at 4°C with the indicated antibodies. After incubation with the corresponding secondary antibody for 1 hour, blots were washed and chemiluminescence was detected with the LAS imager (Fujifilm). Antibodies used in this study were anti-phospho-IRF3 antibody (1:1000, Cell Signalling Technology, CST), anti-VSV G antibody (1:1000, Kerafast), anti-FLAG M2 antibody (1:1000 SIGMA), anti-actin antibody, HRP-linked (1:2000, Santa Cruz), anti-mouse immunoglobulin, HRP-linked (CST), anti-rabbit immunoglobulin, HRP-linked (CST).

As previously described, “total cell lysates were extracted with lysis buffer containing 20 mM HEPES pH 7.5; 1.5 mM KCl; 1 mM EDTA; 1 mM EGTA; 0.15% Triton-X100; 1 mM PMSF; 1 mM DTT; and a cocktail of protease inhibitors (Sigma). The protein content of the lysate was measured using BCA protein assay reagent (Pierce). Aliquots containing an equal amount of protein (30 μg) were separated by SDS-PAGE and then transferred onto PVDF membranes (Sigma). Blots were blocked with 5% non-fat dry milk in PBS Tween 0.1% (PBST) and then incubated with primary antibodies overnight” (30 (link)): anti-phosphoIRF3 antibody (Cell Signaling−4947), anti-phospo-STAT1 (Cell Signaling−9167), anti-STAT1 (Cell Signaling−9172), anti-α-Tubulin (Cell Signaling−2144). Next, blots were incubated with corresponding horseradish peroxidase–conjugated IgG secondary antibody (anti-rabbit or anti-mouse, Cell Signaling). Detection of immunoreactive bands detection was carried out using the enhanced chemiluminescence (ECL) kit (Amersham) according to the manufacturer's instructions.