Anti plin2

Total protein was extracted from C2C12 cells and skeletal muscle tissue using RIPA buffer (Thermo Fisher Scientific) and the mitochondrial fraction was prepared using a mitochondria isolation kit for tissue (Thermo Fisher Scientific) following the manufacturer’s protocol. Protein concentration was measured using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Protein samples were loaded and separated on a 12% SDS PAGE gel and transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad). Non-specific sites were blocked with 5% skim milk in TBST and the membrane was incubated with primary antibodies overnight at 4° C, followed by incubation with HRP-conjugated secondary antibodies for 1 h at room temperature. Antibodies specific to atrogin-1, MuRF1, NCoR1, PGC1α/β, Fkbp5, and PLIN2, as well as a total OXPHOS antibody cocktail were obtained from Abcam (Cambridge, UK). Total and MHC subtype antibodies were supplied by DSHB (IA, USA). Anti-PLIN2 and anti-GAPDH were purchased from Cell Signaling Technologies (MA, USA).