2.6. Detection of phospho-p38 MAPK protein by western blot

Proteins were extracted from gastrocnemius muscle homogenates using ice-cold radioimmunoprecipitation assay (RIPA) buffer supplemented with phosphatase and protease inhibitors. Following centrifugation, proteins were separated by SDS/polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Pierce, Rockford, IL, USA). After transfer, the membranes were washed with PBS then blocked in blocking buffer, followed by incubation overnight at pH 7.6 at 4 °C with antibodies (1:1000, dilution) for p38 MAPK (Cat. No. PA5-17713), phospho-p38 MAPK (Cat. No. 44-684G) and β-actin (Cat. No. PA1-46296) (Thermo Scientific, Rockford, Illinois, USA). After washing, membranes were incubated at 37 °C for 1 h with peroxidase-labeled secondary antibodies (1:4000, dilution). Band intensity was analyzed by ChemiDocTM imaging system with Image LabTM software version 5.1 (Bio-Rad Laboratories Inc., Hercules, CA, USA). Phospho-p38 MAPK protein expression was represented relative to p38 MAPK (as fold change from control).