Sod2 sc 30080

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).

Diaphragm muscle was homogenized 1:10 (w/v) in 5 mM Tris (pH 7.5) and 5 mM EDTA (pH 8.0) with a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) and centrifuged at 1500 g for 10 min at 4 °C. Protein concentration of the resultant supernatant (cytosolic fraction) was assessed by Bradford (Sigma-Aldrich). Proteins were separated via 4–20% gradient polyacrylamide gels containing 0.1% SDS and transferred to nitrocellulose membranes. Membranes were blocked for 2 h at room temperature in PBS solution containing 5% non-fat dry milk and incubated overnight at 4 °C with primary antibodies directed against SOD1 (sc-11407) (Santa Cruz Biotechnology, Dallas, TX, USA), SOD2 (sc-30080) (Santa Cruz), catalase (ab52477) (Abcam, Cambridge, MA, USA), GPXI (ab22604) (Abcam), PRXIII (sc-130336) (Santa Cruz) and α-tubulin (12G10) (DSHB). Appropriate HRP-linked secondary antibodies (anti-rabbit IgG #7074; anti-mouse IgG #7076) (Cell Signaling Technology, Danvers, MA, USA) were diluted in PBS solution containing 0.5% Tween and 5% non-fat milk. Images were acquired via chemiluminescence using Pierce ECL2 substrate (Thermo Fisher Scientific) and the G:Box imaging system (Syngene, Frederick, MD, USA) and were analyzed using ImageJ software (NIH).