Restore plus buffer

Prior to collection, cultured cells were incubated for 30 minutes at 37°C with 10 uM bpVphen (Sigma) in order to inhibit endogenous phosphatases. Cells were then washed twice in PBS and lysed in cell lysis buffer (RIPA buffer, Boston BioProducts; 10% SDS; 100mM Na₃VO₄; 100 mM PMSF; 1X Halt Protease/Phosphatase Inhibitor Cocktail, ThermoScientific). Boiled and clarified lysates were quantitated using DC Protein Assay (Bio-Rad) prior to addition of Laemmli buffer containing 5% β-mercaptoethanol. Equivalent amounts of protein were loaded onto Criterion TGX precast gels (Bio-Rad), which were subsequently transferred to PVDF (Bio-Rad). Membrane were blocked in 5% milk-Tris buffered saline containing 0.05% Tween-20 (TBST), and were incubated with primary antibodies overnight at 4°C at the concentrations indicated in “Reagents and cytokines”. Membranes were washed and incubated for 1 hour with the relevant HRP-conjugated anti-mouse or-rabbit secondary antibody (anti-mouse in all cases except Stat4). Washed membranes were incubated briefly with ECL Reagent (GE Healthcare) and subsequently developed on BIOMAX MR Film (Carestream). For detection of multiple targets on the same membrane, RestorePLUS buffer (Thermo Scientific) was used to strip membranes.