Phospho erk1 2 4376

Cells were lysed with radioimmunoprecipitation assay buffer (RIPA Bufer, Thermo Scientific) containing protease inhibitors (Complete, Mini; Protease Inhibitor Cocktail Tablets, Roche) and phosphatase inhibitors (PhoStop, Phospatase Inhibitor Cocktail Tablets, Roche) and centrifuged at 13000×g, at 4ºC, for 20 minutes. The protein content of the supernatants was determined with BCA assay system (Pierce). Lysate aliquots (50 μg) were resolved by 8%, 10% or 12% (depending on the protein molecular weight) SDS-PAGE and transferred onto nitrocellulose membranes. After blocking with 5% skimmed milk in PBS containing 0.2% Tween 20 at room temperature for 1 hour, membranes were incubated overnight at 4ºC with the appropriate primary antibody (CAV1 #610059 from BD, FoxO1 #2880, ERK1/2 #4695, phospho-ERK1/2 #4376 from Cell Signaling Technology; CAV3 #sc5310 and Myogenin #sc-12732 from Santa Cruz; MyHC MF 20 from Developmental Studies Hybridoma Bank). Blots were then incubated at room temperature for 1 hour with a horseradish peroxidase–conjugated secondary antibody and the peroxidase activity was detected by enhanced chemiluminescence (Pierce) following the instructions of the manufacturer. Immunodetection of β-actin (#ab49900) from Abcam was used as a loading reference.