Anti myosin heavy chain

Immunoblotting was performed as described previously.25 Briefly, cell protein concentration was quantified with the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Following electrophoresis, proteins were transferred on to polyvinylidene difluoride membranes blocked with 5% non‐fat powdered milk and subsequently incubated overnight at 4°C with anti‐Atrogin‐1 (Abcam), anti‐SIRT1, anti‐SIRT3 (Cell Signalling Technology, Danvers, MA, USA), anti‐myosin heavy chain (MHC), and anti‐β actin (Santa Cruz Biotechnology). The signals were detected by using horseradish peroxidase‐conjugated secondary antibodies (Cell Signalling Technology) and revealed with an enhanced chemiluminescence reagent (BioRad Laboratories, Redmond, WA, USA). The gel band quantitative densitometric analysis was quantified by the ImageJ 1.48 software (National Institutes of Health, Bethesda, MD, USA).