Western Blot Analysis of Immune Signaling

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.