Carm1

Cells were seeded on slide covers in 24‐well plates, grown to 80% confluence, and treated as described in 2.10. Then, the cells were fixed with 4% paraformaldehyde for 15 minutes, washed with PBS three times (10 minutes each), permeabilized with 0.25%Triton X‐100 in 3% BSA for 10 minutes, washed with PBS three times, then blocked in 5% BSA at room temperature for 40 minutes. After washing with PBS, the cells were incubated with antibodies against LC3A/B (1:250, Affinity), AMPK (1:200, Affinity), SKP2 (1:200, Affinity), CARM1 (1:250, Affinity) and H3R17me3a (1:200, Affinity) at 4°C overnight. Then, the cells were washed with PBS and incubated with a fluorescent secondary antibody (DyLight 594, E032420, EarthOx, USA; DyLight 488, A23220, Abbkine) in the dark for 60 minutes, stained with DAPI for 10 minutes, then washed twice with PBS. Images were captured using a fluorescence microscope (Eclipse Ti‐S, Nikon, Japan).

C2C12 cells and gastrocnemius muscles were lysed in RIPA buffer, and protein concentrations were determined using a BCA protein assay kit (Thermo Fisher Scientific, USA). Equal amounts of protein were loaded onto 10% SDS polyacrylamide gels, subjected to electrophoresis (at constant pressure, 60 V for 30 minutes, 120 V for 60 minutes), transferred to PVDF membranes (Millipore, Billerica, MA, USA) under a constant current of 350 mA for 120 minutes, blocked with 5% BSA or 5% milk in 1× TBST for 1 hour, then incubated with the following primary antibodies at 4°C overnight: GAPDH (1:2000; EarthOx, USA), MuRF1 (1:1000, Boster, USA), MAFbx (1:1000, Affinity), Myogenin (1:1000, Affinity), LC3A/B (1:800, Affinity), ATG7 (1:800, Affinity), SKP2 (1:1000, Affinity), CARM1 (1:1000, Affinity), H3R17me3a (1:1000, Affinity), AMPK (1:1000, Affinity, AF6423), p‐AMPK (1:1000; Affinity, AF3424), FOXO3a (1:1000, Affinity, AF5418), and p‐FOXO3a (1:1000, Affinity). The membranes were washed with 1× TBST three times (10 minutes each) and incubated with species‐matched HRP‐conjugated secondary antibodies (1:5000, Affinity) for 1 hours, then washed with 1× TBST 3 times (10 minutes each). The blots were visualized with enhanced chemiluminescence (ECL) HRP substrate reagent (WBKLS0500, Millipore, USA) using a MiniChemi610 system (304002L, Beijing, China).