Muscle Atrophy Protein Expression Analysis

GCM and EDL muscles were homogenized in RIPA buffer containing protease/phosphatase inhibitor cocktails and then centrifuged at 12,000 rpm for 1 h at 4°C (Brock Symons et al., 2023 (link)). Proteins (30 µg) were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis to measure their expression. The separated proteins were transferred to a nitrocellulose membrane and then incubated overnight at 4°C with primary antibodies against muscle atrophy, viz. F-box (MAFbx/atrogin-1, ab-168372, 1:1000, Abcam, Cambridge, UK), MuRF1 (ab-172479, 1:1000, Abcam), myosin heavy chain (MyHC, sc-376157, Santa Cruz Biotechnology, CA, USA), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH. Sc-32233; Santa Cruz Biotechnology). After incubation and rinsing with T-BST, the membrane was incubated with an HRP-conjugated secondary antibody for 1 h at room temperature (25°C). Membranes were analyzed using a ChemiDoc XRS+ imaging system (Bio-Rad, Hercules, CA, USA).