Anti p stat4 py693 38 p stat4

Transduced (clover⁺) cells were sorted with a FACSAria cell sorter (BD Biosciences, Dartlab, Dartmouth-Hitchcock Medical Center) and reseeded in fresh media (RPMI 1640 medium supplemented with 10% FBS) for 6 h, prior to culture in the presence or absence of rIL-12 (2.5 ng/ml; 4 h; BioLegend) or pervanadate (0.5 mM Na₃VO₄ and 1.5 mM H₂O₂, mixed 7 min prior to use). Then cells were washed in HBSS, resuspended in ice-cold RIPA buffer with protease and phosphatase inhibitors for 1 h at 4°C, and the lysates were mixed with sample buffer and boiled at 100°C for 5 min. When ready for Western blot assay, equal amounts of protein were loaded onto 10% polyacrylamide gels and probed with purified anti–β-actin (2F1–1) and anti-STAT4 (15A1B41), all from Bio-Legend, and anti–p-STAT4 (pY693; 38/p-Stat4; BD Biosciences). The blots were then incubated with HRP sheep anti-mouse (Jackson ImmunoResearch).