4.5. Procedure for Western Blotting

In this experiment, a conventional western blot system (Bio-Rad Laboratories, Hercules, CA, USA). Proteins in skeletal muscles and adipose tissues were analyzed by electrophoresis USA) was used to analyze specific protein levels using a typical type of electrophoresis. The skeletal muscles and adipose tissues collected were homogenized with CelLytic MT lysis buffer (Sigma-Aldrich, St Louis, MO, USA) mixed with protease inhibitors cocktail (Sigma-Aldrich, St Louis, MO, USA). The total protein concentration was measured by a BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA). An equal amount of proteins was separated by electrophoresis using sodium dodecyl sulphate (SDS)-polyacrylamide gel and then transferred to PVDF membrane. After the membranes were blocked with 5% skim milk, the proteins were incubated with the use of primary antibodies at the given dilutions: β-actin (Santa Cruz Biotechnology, catalog# sc-47778, 1:1000), PGC-1α, (Abcam, catalog# ab-54481, 1:500), FNDC5 (Abcam, catalog# ab-174833, 1:500) and UCP1 (Abcam, catalog# ab-10983, 1:1000). After incubation, wash steps were performed using TBST mixed with tris-buffered saline (TBS) and tween 20 twice for 5 min and twice for 10 min. The membranes were incubated with a secondary antibody (Abcam, 1:2000) for 1 h at room temperature. Thereafter, the wash procedure was performed twice for 5 min and 10 min twice for 30 min. The final band intensity was quantified using a Chemidoc Touch Imaging System (Bio-Rad Laboratories, Hercules, CA, USA) and normalized to that of the corresponding internal reference, β-actin.