Akt and phosphorylated akt

Transfected cells were harvested 48 h after transfection. Cells were lysed in RIPA buffer (Heart, Beijing, China). Total protein samples were separated by SDS‐PAGE polyacrylamide gel and transferred onto a PVDF membrane (Millipore, Billerica, MA, USA). The membranes were immunological overnight at 4°C with primary antibodies as follows: PTEN (1:500; WanleiBio, Shenyang, Liaoning, China); Akt and phosphorylated‐Akt (phosphoresce on S473; 1:500, Abcam, San Francisco, CA, USA); mTOR and phosphorylated‐mTOR (phosphorylated on S2998; 1:1000, Bioworld, Nanjing, Jiangsu, China); Bax and Bcl‐2 (1:1000, Cell Signaling, Danvers, MA, USA); caspase‐9 (1:500, Protein Tech, Wuhan, Hubei, China); and human β‐actin (1:5000; Transgene, China). PVDF membranes were washed with TBST and then incubated with a secondary antibody, HRP‐conjugated goat IgG (1:5000; Transgene, China) for 1 h at 37°C. Signals were detected by the Bio‐Rad Gel imaging system. The images were quantified by Quantity One (Bio‐Rad, USA), and relative protein expression was normalized to β‐actin levels in each sample. All experiments were performed in triplicate.