Anti pgc 1

Immunoblotting was performed as described earlier49 (link) using anti-PIMT (1:1000; Santa Cruz Biotechnology, CA, USA) anti-GLUT4 (1:1000; Novus Biologicals, MO, USA), anti-MEF2A (1:1000; Cell Signaling Technology, MA, USA), anti-PGC-1 (1:1000; Santa Cruz Biotechnology, CA, USA),anti-pSer307-IRS1 (1:1000; Cell Signaling Technology, MA, USA),anti-pTyr608/612-IRS (1:1000; Abcam, MA, USA), anti-IRS1 (1:1000; Cell Signaling Technology, MA, USA), anti-pJNK1/2 (1:1000; Cell Signaling Technology, MA, USA), anti-pAkt (Ser473) (1:1000; Cell Signaling Technology, MA, USA), anti-Akt (1:1000; Cell Signaling Technology, MA, USA) ,anti-ERK1/2 (1:1000; Cell Signaling Technology, MA, USA) and anti-pERK1/2 (1:1000; Cell Signaling Technology, MA, USA) using specific antibodies and appropriate HRP-conjugated secondary antibody. Membranes were stripped and re-probed with β-actin (1:1000, ICN Biomedicals Inc., CA, USA) and Tubulin (1:1000, Genscript, NJ, USA) as the protein loading control.

The skeletal muscle tissues were lysed with liquid nitrogen and RIPA lysis buffer (Beyotime, Jiangsu, China). The lysates were homogenized, and the homogenates were centrifuged at 13,000 g for 15 min at 4°C. Proteins were isolated from cultured cells using RIPA lysis buffer and by following the same protocol. The supernatants were collected, and protein concentrations were determined with a BCA Protein Assay kit (Pierce, number 23225). Equal aliquots (30 μg) of the protein samples were applied to 10% SDS-PAGE gels, transferred to polyvinylidene fluoride (PVDF) membranes, and blocked with 5% skim milk TBST (Tris-buffered Saline Tween-20) buffer. The membranes were incubated with primary antibodies including anti-PGC-1, anti-MnSOD, anti-p53, and anti-GAPDH (1 : 1,000; Santa Cruz Biotechnology) as well as anti-Atg7, anti-p62, and anti-LC3 (1 : 1,000; Cell Signaling) at 4°C overnight. Then, the membranes were incubated with anti-rabbit or anti-mouse antibodies at room temperature for 1 h. The protein bands were captured and documented through a CCD system (Image Station 2000MM, Kodak, Rochester, NY, USA) or a gel image analysis system (ChemiDox XRS, Bio-Rad, USA).