Cu zn sod

Cells were lysed and centrifuged at 13,000 g for 15 min at 4°C. The supernatants were collected, and protein concentrations were determined with the BCA Protein Assay Kit (Pierce). Equal amounts of protein samples were applied to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels, transferred to pure nitrocellulose membranes (PerkinElmer), and blocked with 5% nonfat milk for 2 hours. The membranes were then incubated with the indicated primary antibodies at 4°C overnight. Primary antibodies used in this study were β-actin from Sigma-Aldrich; Atg7, Atg5, Beclin1, and FOXO1 from Cell Signaling Technology; FOXO3 from Sigma-Aldrich; p62, Cu-ZnSOD, MnSOD, and catalase from Santa Cruz Biotechnology; Complex I Ndufs3, Complex I Ndufa9, Complex II subunit 30 kDa Ip, Complex III subunit Core 1, Complex IV Subunit I, and ATP Synthase Subunit Alpha from Invitrogen; and LC3B from Abcam. The membranes were incubated with secondary peroxidase-conjugated antibodies (Jackson ImmunoResearch) at room temperature for 1 h. Chemiluminescent detection was performed by ECL (Pierce). The results were analyzed and quantified by Quantity One Software (Bio-Rad) to obtain the optical density ratio of the target protein to β-actin. One representative result was shown from at least three independent experiments.

Total protein was extracted from frozen muscle tissue using the protein extraction kit (Beyotime Biotechnology, Jiangsu, China) according to the manufacturer’s guide. The protein content of lysates was measured with the Pierce BCA protein Assay kit (Thermo, Waltham, MA, USA). Immunoblotting analysis was performed as previously described [29 (link)]. The primary antibodies included PPARGC1A (ab54481, Abcam, Cambridge, MA, USA), Sirt1 (sc-19857, Santa Cruz Biotechnology, CA, USA), CuZn-SOD (sc-271014, Santa Cruz Biotechnology), and β-tubulin (sc-9104, Santa Cruz Biotechnology). The density of bands was quantified using the Gel Doc XR System (Bio-Rad, Hercules, CA, USA) and then normalized to β-tubulin content. The normalized values were used for comparison of the expression of target protein between SE and LE groups.