P jak2 tyr 1007

Groups of cells were harvested and were lysed with RIPA (Beyotime, Ningbo, China) for 30 min. After centrifugation, the supernatant was collected. After quantification, the protein was denatured, separated with 10% SDS-PAGE by electrophoresis, and transferred to PVDF membranes (Millipore, Bedford, MA, USA). After closure with 5% skim milk powder for 2 h, the membranes were exposed to diluted primary antibodies at 4°C overnight and diluted secondary antibodies (Abcam, Cambridge, MA, USA) for 1.5 h. The protein bands on the membranes were tested by ECL kit, and the protein level was analyzed by graphing. The primary antibodies (p-Stat3 (Tyr 705) and Stat3) were from Abcam (Cambridge, MA, USA); JAK2, p-JAK2 (Tyr 1007), CDK4, Bcl-2, and GAPDH were from Cell Signaling Technology (Beverly, MA, USA); and MMP-9 was from Affinity (JiangSu, China).

Western blot analysis was performed using standard techniques. The following antibodies were used: SHP1, JAK2, p-JAK2 Tyr1007 , STAT3, p-STAT3 tyr705 , and PU.1 (Cell Signaling Technology, Beverly, MA, USA); and Bcl-xl, Bcl-2, cyclin D1, regulatory factor X-1 (RFX-1), and survivin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). As necessary, blots were stripped and reprobed with β-actin antibody (Santa Cruz Biotechnology) as an internal control. Signals were detected with chemiluminescence reagents (Cell Signaling Technology). All experiments were repeated three times with the similar results.