Anti pgc1α ab54481

Protein lysates were obtained by homogenizing paravertebral muscle with lysis buffer. The protein concentration was measured using the Bio-Rad protein assay (Bio-Rad, Richmond, CA, USA). Approximately 50 µg protein was subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes. After blocking with 5% dried milk in Tris-buffered saline (TBS) containing 0.1% Tween-20 for 2 h, the membranes were subsequently incubated overnight with primary antibodies diluted in 5% dried milk-TBS containing 0.1% Tween-20. The primary antibodies were as follows: anti-AMPK (#2532, 1:1,000), anti-phosphorylated (p-)AMPK (:#2531, 1:1,000) (both from Cell Signaling Technology, Danvers, MA, USA), anti-α-tubulin (sc-8035, 1;10,000), anti-β-actin (sc-47778, 1:10,000) (both from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-SIRT3 (ab86671, 1:500), anti-MnSOD (ab13534, 1:2,000) and anti-PGC1α (ab54481, 1:1,000) (all from Abcam Cambridge, MA, USA) antibodies. The membranes were then incubated with appropriate horseradish-peroxidase-conjugated secondary antibodies (sc-2005 and sc-2003, Santa Cruz Biotechnology, Inc.). An enhanced chemiluminescence system was used to visualize the bands. Densitometric analysis was performed using a gel image analysis system (UVP Inc., San Gabriel, CA, USA).