Phospho mtor

Cell lysates were prepared using a kit from Cell Signaling (Beverly, MA, USA). Equal amounts of protein (determined using the Bio-Rad Protein assay) were run on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred to polyvinylidene difluoride membranes, which were washed with Tris-buffered saline (TBS) containing 0.1% Tween 20 (TBST), blocked with TBST and 5% wt/vol nonfat dry milk overnight at 4℃, and incubated with the following primary antibodies: CDK2, β-actin, CyclinD1 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and phospho-mTOR (Ser2448), phospho-mTOR (Ser2481), phospho-p70S6K (Thr389), phospho-4E BP1 (Thr37/46), phospho-AMPKα (Thr172), phospho-Acetyl-CoA carboxylase (Ser79), phospho-extracellular signal–regulated kinase (Thr202/Tyr204), phospho-AKT (Ser473) (Cell Signaling). Membranes were then washed with TBST and incubated with horseradish peroxidase-conjugated secondary antibody (1:1,000). Signals were detected using an Amersham enhanced chemiluminescence (GE Healthcare Life Sciences, Munich, Germany).

After harvesting, the cells were lysed in protein extraction solution (Intron, Daejeon, Korea). Equal amounts of protein were then loaded and separated on a sodium dodecyl sulfate-polyacrylamide gels, and then the proteins were transferred onto nitrocellulose membranes (Pall Corp., Port Washington, NY, USA). After blocking with 5% skim milk, the membranes were incubated with various primary antibodies. The blots were then incubated with peroxidase-conjugated secondary antibody, and enhanced chemiluminescence (Biomax, Seoul, Korea) was used to visualize the specific proteins. The primary antibodies used in western blot analysis were as follows: actin, sterol regulatory element-binding protein-1 (SREBP-1) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), SREBP-2, farnesyl-diphosphate farnesyltransferase-1 (FDFT-1), peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1α, mitochondrial transcription factor A (mtTFA), p62, Parkin (Abcam, Cambridge, UK), fatty acid synthase (FASN), peroxisome proliferator-activated receptor-γ (PPAR-γ), insulin-like growth factor-1 receptor (IGF-1R), phospho-IGF-1R, Akt, phospho-Akt, mechanistic target of rapamycin (mTOR), phospho-mTOR, (Cell Signalling Technology, Danvers, MA, USA), stearoyl-coenzyme A desaturase (SCD) (Thermo Scientific, Rockford, IL, USA), and LC-3 (Sigma, St. Louis, MO, USA).