The assay was performed as described previously [26 (link)]. Cells were processed using radioimmunoprecipitation assay (RIPA) buffer, and the obtained cell lysate was centrifuged at 12,000 rpm for 20 min at −4°C. The cell lysate supernatant was collected for quantification using a bicinchoninic acid (BCA) kit (BCA Protein Assay Kit, P0010). Protein samples (25 µg) were fractionated using 10% sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) before polyvinylidene fluoride (PVDF) membrane transfer. Primary antibodies were diluted as instructed and then incubated with the membrane in a cool room for 24 h, followed by continuous incubation with horseradish peroxidase (HRP)-coupled secondary antibody at room temperature for another 1 h. Pierce electrogenerated chemiluminescence (ECL) Western blot substrate (Thermo Scientific, USA) was used to develop the membrane on X-ray films. The antibodies used were as follows: anti PI3K (ab278545), anti-Akt (ab38449), anti-phosphorylated (p)-Akt (ab38449), anti-p-mTOR (ab137133), and anti-mTOR (ab134903) antibodies obtained from Abcam (Cambridge, United States).