Western Blotting Analysis of Signaling Pathways

Cell lysates were prepared and western blotting was performed as described previously (42 (link)). Proteins were separated using 6%, 10%, or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were detected using the following primary antibodies: anti-phospho-AMPKα (Cell Signaling Technology, clone 40H9), anti-AMPKα (Cell Signaling Technology), anti-phospho-mTOR (Cell Signaling Technology, clone D9C2), anti-mTOR (Cell Signaling Technology, clone 7C10), anti-β-actin (Cell Signaling Technology, clone 13E5), anti-DDIT4 (Proteintech), anti-phospho-Rictor (Cell Signaling Technology, clone D30A3), anti-Rictor (Cell Signaling Technology, clone 53A2), anti-phospho-FoxO3a (Cell Signaling Technology, clone D18H8), anti-FoxO3a (Cell Signaling Technology, clone D19A7), anti-phospho-AKT (Cell Signaling Technology, clone Ser473), anti-AKT (Cell Signaling Technology, clone Ser473), anti-phospho-p70 S6 Kinase (Thr389) (Cell Signaling Technology, 9205S), anti p70 S6 Kinase (Cell Signaling Technology.9202S), anti-phospho-Raptor(Ser792) (Cell Signaling Technology, 2083S), and Raptor (24C12) (Cell Signaling Technology.2280S) antibodies. After the incubating with the corresponding secondary antibody, the ECL substrate (Thermo Fisher) signals were detected using an Amersham Imager 600 (Cytiva Life Sciences, GE HealthCare).