Immunoblot analysis of STAT1 phosphorylation.

Mouse IFN-γ-treated BMDMs and MEFs were lysed in radioimmunoprecipitation assay (RIPA) buffer (catalog no. 9806S; CST) supplemented with a protease inhibitor (catalog no. 78430; Thermo Fisher) and phosphatase inhibitor (catalog no. 88667; Thermo Fisher). An equal amount of protein was loaded and separated on 10% SDS-PAGE gels (Bio-Rad), and then transferred to polyvinylidene fluoride membranes (EMD Millipore). Primary antibodies against phosphor-STAT1 (Tyr701), STAT1, and GAPDH were stained and detected by the use of horseradish-peroxidase-conjugated secondary anti-rabbit antibody (catalog no. 31460; Invitrogen). All blots were performed using Pierce ECL substrate (Thermo Fisher Scientific) and scanned with a ChemiDoc touch imaging system (Bio-Rad).