Western blot

Total protein extraction was carried out using RIPA solution (Thermo Fisher Scientific) and the BCA assay was performed to measure protein concentration. After denaturing, protein samples (20 μg from each sample) were subjected to 12% SDS-PAGE gel electrophoresis, followed by gel transfer to PVDF membranes. Blocking was performed by incubating membranes with 5% skimmed milk for 1 h at room temperature.

After blocking, the membranes were incubated with rabbit anti- human primary antibodies of p-Akt (1:2000, ab38449, Abcam), Akt (1:2000, ab8805, Abcam) and GAPDH (1:1000, ab8245, Abcam) at 4 °C overnight. After that, membranes were washed 3 times for 5 min each time with phosphate-buffered saline (PBS). The membranes were further incubated with anti-rabbit IgG-HRP secondary antibody (1:1000, MBS435036, MyBioSource) at room temperature for 2 h. Finally, ECL (Sigma-Aldrich) was added and a MYECL Imager (Thermo Fisher Scientific) was used to detect signals. The relative expression levels of p-Akt and Akt were normalized to the endogenous control GAPDH using Image J software.