Ab199010

Western blots were performed and analyzed as previously described [22 (link)]. Briefly, gastrocnemius muscle samples were homogenized in RIPA buffer, and then centrifuged at 12000 × g for 30 min at 4 °C. The quantification of protein in supernatant was accomplished using a BCA kit (Beyotime Biotechnology, Wuhan, China). The protein samples were boiled in the presence of sample buffer at 95 °C for 3 min. The proteins were subjected to the separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and subsequently transferred onto a nitrocellulose membrane. The target protein was probed by a corresponding antibody and then visualized by enhanced chemiluminescence (ECL) reagent and imaged by the chemiluminescence imaging system Amersham Imager 680 (General Electric Company, USA). Antibodies used for Western blots were: Anti-Pax7 (Abcam, USA, ab199010), Anti-alpha smooth muscle Actin (Abcam, USA, ab5694), Anti-LAMP2-Lysosome Marker (Abcam, USA, ab25631), Anti-Beta actin (Abcam, USA, ab8226), Anti-Cleaved LC3B (Sigma, Germany, L7S43), Anti-ARPC5/p16 ARC (Abcam, USA, ab51243), p53 (1C12) Mouse mAb (Cell Signaling Technology, USA, 1C12). Origin images of all western blot have been uploaded as a single ‘Supplemental Material’ file.

cDNA was synthesized in accordance with the instructions provided in the reverse transcription kit (Vazyme, Shanghai, China). SYBR, diethyl pyrocarbonate water, cDNA, β‐actin as the reference gene, and upstream and downstream primers were mixed proportionally to 10 μL for the polymerase chain reaction. The primer sequences are shown in Table S3.
After the concentration was determined by the BCA method, SDS/PAGE electrophoresis was performed and the target protein was transferred to the nitrocellulose membrane. The membrane was incubated with primary antibody overnight at 4 °C, followed by secondary antibody for 1 h at 37 °C. The protein was visualized with a gel imager (Bio‐Rad, Hercules, CA, USA). The primary antibodies included Pax7 and anti‐MyoD (ab199010 and ab16148; Abcam); PPARγ and Glyceraldehyde 3‐phosphate dehydrogenase (16643‐1‐AP and 60004‐1‐AP; Proteintech); α‐smooth muscle actin (α‐SMA; 19245, Cell Signaling, Danvers, MA, USA); Collagen‐1 (ab255809; Abcam); anti‐β‐actin (CW0096M; CWBIO), and TSG101 and CD9 (28283‐1‐AP and 20597‐1‐AP; Proteintech).

Primary antibodies used against proteins were the following: α-tubulin (sc-23948, Santa Cruz, Texas, USA); ATG7 (8558, Cell signaling, Danvers, MA); Beclin-1 (4445, Cell signaling); CI-20 (NDUFB8) (ab110242, Abcam, Cambridge, UK); CII-30 (SDHB) (ab14714, Abcam); CIII-Core II (UQCRC2) (ab14745, Abcam); CIV-I (MTCO1) (ab14705, Abcam); CV-a (ATP5A) (ab14748, Abcam); cytochrome C (ab110252, Abcam); LC3 (PD014, Medical & Biological Laboratories Co., LDT, Japan); MYF5 (ab125078, Abcam); MYOD (sc-760, Santa Cruz); Myogenin (ab1835, Abcam); p16 lnK4a (1661, Santa Cruz Biotechnology); PAX3 (MAB2457, R&D System, MN, USA); PAX7 (ab199010, Abcam); phospho-SHC/p66 (566807, Calbiochem); PGC-1α (ab3242, Millipore); porin (MSA03) (ab14734, Abcam); SHC (610879, BD Trasduction Laboratories); SQSTM1/p62 (H00008878-M01, Abnova, Taipe, Taiwan); TFAM (PA5-29571, Thermo Fisher, USA) and TOM20 (5490, Cell Signallin).
Secondary antibodies used were the following: goat anti-rabbit IgG secondary antibody (7074 S, Cell Signaling Technology); horse anti-mouse IgG secondary antibody (7076 S, Cell Signaling Technology).