Western blot analysis

Cells were lysed with RIPA buffer (Beyotime Institute of Biotechnology) containing protease inhibitors. Protein concentration was quantified using bicinchoninic acid protein assay (Pierce; Thermo Fisher Scientific, Inc.). Protein samples (10 µg) were separated on 12% SDS-PAGE gel and transferred to polyvinylidene fluoride membranes (Roche, Basel, Switzerland). Membranes were incubated in 5% non-fat milk at 25°C for 1 h to block non-specific binding. Membranes were then membranes incubated overnight at 4°C with primary antibodies targeting phosphorylated (p-) Akt (cat. no. ab81283; 1:500; Abcam, Cambridge, MA, USA), total Akt (cat. no. ab18785; 1:500; Abcam), and β-actin (cat. no. ab8226; 1:500; Abcam). Membranes were washed three times with PBS, then samples were incubated with anti-rabbit or anti-mouse secondary antibodies purchased from Beyotime Institute of Biotechnology (cat. no. A0208/A0216; 1:1,000). Bands were visualized using enhanced chemiluminescence substrate (Thermo Fisher Scientific Inc.). Quantity One (version 4.0; Bio-Rad Laboratories, Inc.) was used for densitometric analysis.