Western blotting

Cells were lysed in RIPA protein extraction reagent supplemented with a protease inhibitor cocktail (Roche, CA, USA) and phosphatase inhibitor cocktail (Roche). Protein concentration was measured using a bicinchoninic acid assay (BCA). Equal amounts of total protein lysate were separated by 10% SDS-PAGE and transferred onto PVDF membranes. After blocking in 5% non-fat milk, the membranes were probed with targeted primary antibodies overnight at 4°C and detected by incubating with specific secondary antibodies for 2 h at room temperature. ECL chromogenic substrate with HRP was used to visualize the protein bands, and the intensities of the bands were quantified using Image J software. GAPDH served as an internal loading control. Primary anti-human antibodies against total Akt, Erk1/2, P38, and JNK and phosphorylated-Akt, Erk1/2, P38, and JNK were purchased from Cell Signaling Technology (MA, USA).