Description
Rat skeletal muscle tissue lysate was prepared by homogenization in modified RIPA buffer (150 mM sodium chloride + 50 mM Tris-HCl, pH 7.4 + 1 mM ethylenediaminetetraacetic acid + 1 mM phenylmethylsulfonyl fluoride + 1% Triton X-100 + 1% sodium deoxycholic acid + 0.1% sodium dodecylsulfate + 5 µg/ml of aprotinin + 5 µg/ml of leupeptin). Tissue and cell debris was removed by centrifugation. Protein concentration was determined with Bio-Rad protein assay. The tissue lysate was boiled for 5 min in 1 x SDS sample buffer (50 mM Tris-HCl, pH 6.8 + 12.5% glycerol + 1% sodium dodecylsulfate + 0.01% bromophenol blue) containing 5% β-mercaptoethanol