Phospho stat1 ser727

Naive CD4 1 T cells were cultured in RPMI 1640 medium with 10% FBS. After stimulation, cells were washed once in cold PBS and lysed in RIPA buffer (50 mmol/L Tris-HCl [pH 7.4], 1% NP-40, 0.25% Na-deoxycholate, 150 mmol/L NaCl, and 1 mmol/L EDTA [pH 7.4]) with protease and phosphatase inhibitor cocktails (Roche, Mannheim, Germany) for 10 minutes on ice. Protein concentrations were determined by using a BCA assay. 38 The lysates were mixed with 53 SDS-PAGE loading buffer and boiled at 1008C for 5 minutes. Protein samples were size fractionated on 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Millipore, Temecula, Calif). After blocking for 1 hour, membranes were incubated with primary antibodies overnight at 48C. The appropriate horseradish peroxidase-conjoined secondary antibody was then added and detected by using chemiluminescence (Millipore). Actin was used as a protein loading control. Phospho-STAT6 (Tyr641), STAT6, phospho-Smad3 (Ser423/425), Smad3, phospho-p38MAPK (Thr180/Tyr182), P38, phospho-STAT5 (Tyr694), STAT5, phospho-STAT1 (Ser727), STAT1, phospho-SAPK/JNK (Thr183/Tyr185), SAPK/JNK, phospho-c-Jun (Ser73), c-Jun, Rel-B, NF-kB p65, b-actin, and histone H3 were purchased from Cell Signaling Technology (CST, Danvers, Mass).