Cell Lysate Preparation and Western Blots

Cells were lysed by RIPA buffer (100 mM Tris-HCl, 150 mM NaCl, and 0.1% SDS, and 1% Triton-X-100) at 4°C for 10 min, and the cell lysates were harvested by centrifugation at 15,000 rpm for 10 min to obtain the supernatants. Twenty-microgram cell lysates from each group were applied to 8% and 12% sodium dodecyl sulfate polyacrylamide gels electrophoresis. Proteins were transferred onto polyvinyl difluoride membranes (Millipore, MA, USA) and blocked with 5% skim milk in TBST for 1 h at room temperature. The antibodies used are ACTN (Santa Cruz Technology Dallas, TX, USA; SC-17829), p-mTOR (Cell Signaling Technology, Danvers, MA, USA; CST-2971), mTOR (Cell Signaling Technology, Danvers, MA, USA; CST-2972), p-AKT (Cell Signaling Technology, Danvers, MA, USA; CST-9271), AKT (Cell Signaling Technology, Danvers, MA, USA; CST-4691), p-ERK (Cell Signaling Technology, Danvers, MA, USA; CST-4370), ERK (Cell Signaling Technology, Danvers, MA, USA; CST-4695), p62 (Santa Cruz Technology Dallas, TX, USA; SC- 28359), LC3B (Cell Signaling Technology, Danvers, MA, USA; CST-2775), BCL2 (Cell Signaling Technology, Danvers, MA, USA; CST-4223), p-CDC2 (Thr15) (Cell Signaling Technology, Danvers, MA, USA; CST-9111) and CDC2 (Cell Signaling Technology, Danvers, MA, USA; CST-9112). Band detection was conducted by enhanced chemi-luminescence and X-ray film (GE Healthcare, Piscataway, NJ, USA).