P src

Cells after treatment were lysed and centrifuged at 12,000 × g for 15 min. The supernatant was collected and the protein concentrations were detected using the BCA assay kit (Beyotime, Jiangsu, China). Equal amounts of total proteins (20–60 μg) were separated using SDS-polyacrylamide gel and electrotransferred to PVDF membranes. The membranes were initially blocked with 5% nonfat dry milk in PBS-Tween-20 (0.1%, v/v) at 4°C for 12 h and then probed with primary antibodies against cleaved caspase-3 (mouse IgG1), Bcl-2 (mouse IgG1 κ), Bax (mouse IgG1 κ), STAT3 (mouse IgG1 κ), p-STAT3 (mouse IgG1), p-JAK2 (Rabbit mAb), JAK2 (mouse IgG2b κ), p-Src (mouse IgG2a κ), Src (mouse IgG2a κ) or GAPDH (Rabbit mAb) (Santa Cruz, CA, United States), which were diluted following the manufacturer’s instructions at 4°C overnight. Then the membranes were blotted with the horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (Beyotime, Jiangsu, China, 1:5,000) for 2 h at 37°C. Finally, the bands were visualized through the enhanced chemiluminescence protocol. Signals were densitometrically quantified and normalized to GAPDH. The results were analyzed using Image J software (NIH, United States).