Pgc1α sc 13067

Immunoblotting was performed as described previously [7 (link)]. The following specific primary antibodies were used against p16 (sc-74400, 1:200; sc-1661, 1:500), SOD1 (sc-11407, 1:500), SOD2 (sc-30080, 1:200), ND4 (sc-20499-R, 1:200), PGC-1α (sc-13067, 1:200), PGC-1β (sc-373771, 1:200), PRC (sc-135516, 1:200), TFAM (sc-166965, 1:200), and Rb (sc-102, 1:500) obtained from Santa Cruz Biotechnology (Santa Cruz, CA). When the p16 antibodies were no longer available from Santa Cruz, we used a different antibody (NA29, 1:100) from EMD Millipore (Billerica, MA). The β-actin antibody was used at a 1:1000 dilution and obtained from Sigma-Aldrich (A-3853, St. Louis, MO, USA). The ATP5A (ab14746, 1;200), UQCRC2 (ab14745, 1:200), and tubulin (ab21058, 1:5000) antibodies were obtained from Abcam (Cambridge, MA). The SDHA antibody (MS 204M) was used at a 1:200 dilution and obtained from Mitoscience (Eugene, OR). The VDAC antibody (PAI-954A) was used at a 1:200 dilution and obtained from ABR Affinity Bioreagents (Golden, CO). The phospho-Rb (8180S, 1:1000), CDK4 (12790S, 1:1000), and CDK6 (3136S, 1:500) antibodies were obtained from Cell Signaling Technology (Beverly, MA).

For protein content analysis, skeletal (quadriceps femoris) and cardiac (left ventricle) muscle samples were homogenized and analyzed by Western blotting. The following antibodies were used: BNIP3 (ab109362, rabbit, 1 : 1000), p-AMPK (ab131357, rabbit, 1 : 500) from Abcam (USA); PGC-1α (sc-13067, rabbit, 1 : 200) from Santa Cruz (USA); CS (16131-1-AP, rabbit, 1 : 1000), LC 3 I/II (14600-1-AP, rabbit, 1 : 500), AMPK (10929-2-AP, rabbit, 1 : 500), DRP1 (12957-1-AP, rabbit, 1 : 500), MFN1 (13798-1-AP, rabbit, 1 : 500), and p62 (18420-1-AP, rabbit, 1 : 1000) from Proteintech (USA); and GAPDH (AP0063, rabbit, 1 : 5000) from Bioworld (USA). Membranes were analyzed and quantified using the Quantity One Imaging System (BIO-RAS, USA). Protein expression was normalized to that of GAPDH.

Total protein was isolated from cells or tissues using RIPA buffer. Protein separation and western blot analysis were conducted as described earlier^{66 (link)}. GADD45α antibody (GTX54090, 1:1000) was from GeneTex. UCP1 (ab10983, 1:2000) and Perilipin (ab61682, 1:2,000) were from Abcam. FABP4 (E71703-98, 1:2000), GAPDH (EM1101, 1:5000) was from HuaBio. PPARγ (C26H12, 1:1000) was from Cell Signaling Technology (CST). Cocktail (45-8099, 1:2000) is from Thermo Fisher Scientific. PGC1α (sc-13067) was from Santa Cruz Biotechnology (Santa Cruz). The horseradish peroxidase (HRP)-conjugated secondary antibody (anti-rabbit IgG, 111-035-003 or anti-mouse IgG; 115-035-003, Jackson ImmunoResearch) was diluted 1:10,000. Immunodetection was performed using enhanced chemiluminescence western blotting substrate (Google Biotechnology, Wuhan, Hubei, China) and detected by ChemiScope3500 Mini System.