Pgc 1α

Muscle samples were lysed and protein concentration was determined as previously described (26 (link)). Immunoblotting was performed using the following primary antibodies: pAkt Ser⁴⁷³, pAkt Thr³⁰⁸, total Akt, pSAPK/JNK Thr¹⁸³/Tyr¹⁸⁵, total JNK, SIRT1 (mouse specific), acetylated Lys, parkin, LC3, ATG12, PKC-α, PKC-δ, and PKC-θ (Cell Signaling); lipoprotein lipase (LPL) (Abcam); CD36 (R&D Systems); p62 (ProGen); PINK1 (Cayman Chemicals); PGC-1α (Millipore); and α-tubulin (Sigma-Aldrich). For protein kinase C (PKC) studies, subcellular fractionation was carried out as previously described (13 (link)).

Western blots were performed on lysates prepared from tissues collected at the end of the experiments. Samples of 30 μg of protein were boiled in Laemmli sample buffer (Bio-Rad, Hercules, CA) for 5 min and electrophoresed on 10 or 12.5% SDS-polyacrylamide gels and blotted onto nitrocellulose membrane. Membranes were blocked with Odyssey blocking buffer (LI-COR, Lincoln, NE) for 2 hours at room temperature and then incubated with primary antibodies overnight at 4°C. Membranes were incubated with either Alex 680 (Molecular Probes) or IRDye 800 (Rockland, Gilbertsville, PA) secondary antibodies for 1 hour at room temperature. Membranes were visualized using an Odyssey infrared imager (Li-COR, Lincoln, NE) which allows for the simultaneous detection of 2 proteins. Densitometry analysis was performed using Odyssey software (LI-COR, Lincoln, NE). Antibodies for Western blots were as follows: HMBG1 (Abcam, Boston, MA), NRF-1 (Rockland, Gilbertsville, PA), PGC1-α (Millipore, Temecula, CA), UCP-1 (Sigma, St. Louis, MO) and β-actin (Abcam, Cambridge, MA). All antibodies were used at a ratio of 1:1000 with blocking buffer, the lone exception being β-actin which was used at a ratio of 1:5000. All blots from tissue samples were run with 3 samples from all groups of mice per gel.