Phosphor stat5

The tumor tissues were surgically excised and frozen in liquid nitrogen and then homogenized in tumor lysis buffer (Prod# 78510, Thermo, USA); after centrifugation at 12,000 g for 10 min at 4°C, the lysates were collected. The protein was quantified using a BCA Kit (Prod# 23225, Thermo, USA), separated on SDS-PAGE gels at 8%–14% polyacrylamide according to protein weight and blotted onto a PVDF nitrocellulose membrane (Bio-Rad Laboratories, USA). The membrane was blocked in 5% milk in PBST for 1 h and then probed with primary antibodies overnight at 4°C. The following primary antibodies were used: the phosphor-EGFR, EGFR, phosphor-ERK, ERK and Bcl-xL antibodies, which were purchased from Santa Cruz Biotechnology, and the mTOR, phosphor-mTOR, phosphor-4EBP1, 4EBP1, p70S6K, phosphor-p70S6K, cleaved caspase-3, caspase-3, PARP, phosphor-AKT, AKT, Jak1, phosphor-Jak1, STAT5, phosphor-STAT5, STAT3 and phosphor-STAT3 antibodies, which were obtained from Cell Signaling Technology. After washing the membranes in PBST, they were incubated with the appropriate secondary antibodies for 1 h at room temperature, washed three times in PBST and then visualized with enhanced chemiluminescence reagent, following the manufacturer's instructions (Prod# 34080, Thermo, USA).