Muscle Fiber Protein Extraction and Analysis

The muscle fibers were solubilized with radio-immunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate) including protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 mM cocktail of protease inhibitors) and scraped from the plates to prepare cell extracts. The protein concentrations were determined using a micro-BCA assay. Protein extracts from muscle fibers incubated with DCA, CA, or vehicle were obtained, mixed with loading buffer, and heated to 50 °C for 5 min. Furthermore, the samples were subjected to 12% SDS-PAGE. Electrophoretic transference was applied to polyvinylidene difluoride (PVDF) membranes (Thermo Fisher Scientific, Waltham, MA, USA). The membranes were blocked with 5% skim milk–Tris buffer saline (TBS) and incubated separately with anti-OXPHOS (1:1000; Abcam, Cambridge, MA, USA) and anti-β-actin (1:1000; Abcam, Cambridge, MA, USA) antibodies. The chemiluminescent signals (Thermo Scientific, Waltham, MA, USA) were detected using secondary antibodies and peroxidase-coupled IgG (1:1000) with Fotodyne (Fisher Scientific, St. Waltham, MA, USA). The blots were then quantified by densitometry using ImageJ software (NIH, Bethesda, MD, USA) [17 (link)].