Cleaved casp3

Total protein from homogenized tissues or cell lysates was extracted using ice-cold RIPA solution (Fudebio, China) and protease inhibitors (Fudebio, China). The protein samples were diluted to equal concentrations, denatured in a boiling water bath, separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes. Membranes were blocked with 5% nonfat milk in Tris-buffered saline-Tween (TBST) buffer and were then incubated overnight at 4 °C with primary antibodies against cleaved CASP3 (1:1000) (Affinity Biosciences, USA), CCND1 (1:1200) (Proteintech, China), CDK4 (1:2000) (Abcam, USA), BAX (1:1000) (Abcam, USA), BCL2 (1:800) (Abcam, USA), E-cadherin (1:2000) (Proteintech, China), N-cadherin (1:2000) (Proteintech, China), Vimentin (1:3000) (Proteintech, China), FGF2 (1:200) (Santa Cruz Biotechnology, USA), PI3K (1:1000) (Cell Signaling Technology, USA), phosphorylated PI3K (p-PI3K, 1:1000) (Cell Signaling Technology, USA), Akt (1:1000) (Cell Signaling Technology, USA), phosphorylated Akt (p-Akt, 1:1000) (Cell Signaling Technology, USA) and GAPDH (1:10000) (Proteintech, China). After four washes with TBST buffer, membranes were incubated for 60 min at 25 °C with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10000) (Bioss, China). Protein bands were visualized with a chemiluminescence imaging system (Bio-Rad, USA).