Frozen tissues from 4- to 7-month-old mice were lysed in RIPA lysis buffer [50 mM tris-HCl (pH 8), 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 0.1% SDS, and 0.5% deoxycholic acid], supplemented with a cocktail of protease and phosphatase inhibitors (Pierce), using a Precellys homogenizer (Bertin). Protein concentration was assessed using the bicinchoninic acid assay (Pierce). Protein extracts (20 μg) from superficial gastrocnemius muscles of 6-month-old WT and Hacd1-KO mice were loaded on Bolt 4 to 12% bis-tris gels (Invitrogen), separated for 22 min at 200 V and subsequently transferred to polyvinylidene difluoride membranes using transfer stack and iblot2 system for 7 min at 20 V (Invitrogen). Thereafter, blots were blocked for 60 min in 1.25% gelatin in tris-buffered saline with Tween 20 at room temperature, followed by incubation with different primary antibodies [phospho-AMPK, AMPK, and CS from Cell Signaling; ATP5A from Abcam; VDAC (custom-made gift from C. Lemaire, INSERM U1180)], overnight at 4°C. After washing, membranes were incubated with the appropriate secondary antibodies for 60 min at room temperature and revealed using the West Femto chemiluminescent substrate (Pierce). Light emission was recorded using a chemiluminescent detection system (G-Box, Syngene) and quantified by the ImageJ software [v1.47, National Institutes of Health (NIH)]. Protein content was normalized to β-actin (Antibody from Santa Cruz Biotechnology).

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