Anti pgc 1α

PGC-1α lysine acetylation was analyzed by immunoprecipitation of PGC-1α followed by western blot using acetylated-lysine antibody (1:1000; Cell Signaling, 9681S, MA, USA). Tissue protein extracts and protein content were prepared using RIPA buffer and BCA protein assay kit. For immunoprecipitation, 500 μg of protein lysates samples were incubated at 4°C rotation overnight with anti-PGC1α (2 μg; Santa Cruz, SC-13067, CA, USA) diluted as recommended in the manufacturer's instructions. Purified proteins were separated by 8% SDS-PAGE and immunoblotting using anti-PGC1α (1:200; Santa Cruz, CA, USA) and acetylated-lysine (1:1000; Cell Signaling Technology, USA). The supernatant and precipitated protein was immunoblotted with PGC-1α and actin antibody to confirm the immunoprecipitation process (S2 Fig).

Cells were lysed using ice-cold lysis buffer (0.1 mL of 50 mM Tris-HCl (pH 7.2) containing 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, and 1% NP-40), and the so-obtained cell lysates were assayed for protein concentration by Bradford staining. Equal amounts of protein (20 μg/mL) were electrophoresed on 10% SDS-acrylamide gels and transferred to nitrocellulose membranes using an electric transfer system. Nonspecific binding was blocked by treating membranes with 3% skim milk in TBST buffer (5 mM Tris-HCl, pH 7.6, 136 mM NaCl, and 0.1% Tween-20) for 1 h. Blots were incubated for 1 h at room temperature with primary antibody against anti-phospho-AMPKα (Thr 172), anti-AMPKα, anti-phospho-acetyl-CoA carboxylase (ACC) (Ser79), anti-ACC, anti-SIRT1 (Cell Signaling Technology, Danvers, MA, USA), anti-PGC1α (Santa Cruz Biotechnology, Santa Cruz, CA, USA), glucose transporter (GLUT) 4 (Santa Cruz Biotechnology), and anti-β-actin (Sigma-Aldrich, St. Louis, MO, USA) and then incubated for 1 h at RT with horseradish peroxidase- (HRP-) labeled anti-mouse IgG (1 : 1000, Santa Cruz Biotechnology), washed with 1x TBST three times, and developed with the ECL Western detection reagents (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). Protein bands were quantified by densitometry using Image J.