Western Blot Analysis of Cellular Proteins

Cell and tissues lysates were collected as previously described (29 (link)). Protein concentrations were determined using BCA Assay (Beyotime Biotechnology). Equal amounts of protein were separated with 8–12% SDS-PAGE and then electrophoretically transferred onto a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). TBST containing with 5% non-fat milk or bovine serum albumin was used to block non-specific binding for 2 h at room temperature. Then, membranes were incubated with primary antibodies according to the instructions overnight at 4°C followed by appropriate secondary antibodies. Signals generated by enhanced chemiluminescence (Millipore) were recorded with a CCD camera (Tanon, Shanghai). The experiments were performed three biological repeats. Primary and secondary antibodies included: ALDH1A3 (1:1,000, Sigma-Aldrich, #HPA046271), HK2 (1:1,000, Cell Signaling Technology, #2867), PKM2 (1:1,000, Cell Signaling Technology, #4053), PPARγ (1:1,000, Santa Cruze, #sc-7273), mTOR (1:1,000, Cell Signaling Technology, #2983), p-mTOR (1:1,000, Cell Signaling Technology, #5536), PI3 Kinase p110α (1:1,000, Cell Signaling Technology, #4249), Akt (1:1,000, Cell Signaling Technology, #2920), p-Akt (1:1,000, Cell Signaling Technology, #4060), β-actin (1:5,000, Sigma-Aldrich, #5441).