Muscle Protein Expression Analysis

Muscle SREBP-1c, ACLY, LXRs, FAS, MAFbx, MuRF-1, MyoD, Myostatin, IL-1β, TNF-α, and NCAM levels were determined using the Western blot technique [27 (link)]. The muscle homogenates were prepared in ice-cold lysis buffer. SDS-PAGE sample buffer containing 2% β-mercaptoethanol was added to the supernatant. Twenty micrograms of protein were electrophoresed and then transferred into Nitrocellulose membranes (Schleicher and Schuell Inc., Keene, NH, USA). Nitrocellulose blots blocked with 1% bovine serum albumin in PBS for one hour prior to administration of the primary antibodies (SREBP-1c, ACLY, LXRs, FAS, MAFbx, MuRF-1, MyoD, Myostatin, TNF-α, IL-1β, and NCAM) (Abcam, Cambridge, UK) that were diluted (1:1000) in the same buffer containing 0.05% Tween-20. Protein loading was checked using an antibody against α-actin (A5316; Sigma Aldrich, St. Louis, MO, USA). Bands were analyzed densitometrically using an image analysis system (Image J; National Institute of Health, Bethesda, MA, USA).