Pw0034

The acquired gastrocnemius tissues were lysed with the assistance of a lysis buffer (abs9229, absin, China). Subsequently, we selected the BCA kit (BI-WB005, SBJBIO, China) to quantify the lysed protein. Thereafter, the quantified protein was electrophoresed for protein separation, which was then loaded onto the PVDF membrane (PW0034, Leagene, China). After being sealed in 5% bovine serum albumin (BL-082, SBJBIO, China) at 37°C for 60 min, the membrane underwent primary antibodies (4°C, overnight). Afterward, the bound antibodies were then exposed to anti-rabbit secondary antibody (31466, Invitrogen, USA) or anti-mouse secondary antibody (S0002, Affinity, USA) with the condition of 37°C for 60 min. Visualization of protein was conducted by applying an ECL reagent (GK10008, GlpBio, USA) on a gel imaging system (A44114, Invitrogen, USA). The primary antibodies of PGC-1α (1:1,000, ab191838), P62 (1:10,000, ab109012), LC3B (1:2,000, ab48394), AMPKα (1:5,000, ab32047), p-AMPKα (1:10,000, ab133448), Smad3 (1:1,000, ab208182), p-Smad3 (1:2,000, ab52903), and β-actin (1:5,000, ab8227) were provided by Abcam (UK).

For western blot analysis, proteins were extracted from femoral tissues using RIPA Buffer (20101ES60, Yeasen, China). We used the BCA kit (BI-WB005, SBJBIO, China) for protein quantification. After electrophoresis, the protein was loaded onto PVDF membranes (PW0034, Leagene, China). Next, 5% bovine serum albumin (BL-082, SBJBIO, China) was added to seal the membranes (37 °C, 60 min). The membranes were then immersed in primary antibodies (4 °C, overnight) and subsequently in anti-rabbit secondary antibodies (31,466, Invitrogen, USA) or anti-mouse secondary antibodies (S0002, Affinity, USA) at 37 °C for 60 min. Protein visualization was performed using an ECL reagent (GK10008, GlpBio, USA) on an eZwest Lite Auto Imaging System (Genscript, USA). The primary antibodies of Nrf2 (1:2000, AF0639), heme oxygenase 1 (HO-1) (1:2000, AF5393), phospho-IKB alpha (Ser32/Ser36, 1:2000, AF2002), IKB alpha (1:2000, AF5002), phospho-NF-kB p65 (Ser536, 1:2000, AF2006), NF-kB p65 (1:2000, AF5006), phospho-extracellular regulated protein kinases ½ (ERK1/2) (Tyr204, 1:2000, AF1014), ERK1/2 (1:2000, AF0155), phospho-p38 mitogen-activated protein kinase (MAPK) (Thr180/Tyr182, 1:500, AF4001), p38 MAPK (1:1000, AF6456), and GAPDH (1:20,000, AF7021) were obtained from Affinity (USA). GAPDH was used as the loading control.