Anti chd4

Protein lysates from MuSC were obtained using protein lysis buffer I [66mM Tris-HCl and 2% SDS] supplemented with protease and phosphatase inhibitors (0.5 μg/ml benzamidine, 2 μg/ml aprotinin, 2 μg/ml leupetin, 2mM PMFS, 1mM Na3Vo4, 20mM NaF). Whole muscle cell lysates were generated by finely mincing isolated muscle tissue and resuspended in protein lysis buffer II (100 mM Tris-HCl, 10% SDS and 12.7 mM EDTA) supplemented with protease and phosphatase inhibitors as described above. Protein lysates were sonicated and clarified by centrifugation at 14000 rpm for 15 min. Protein concentrations were determined with DC Protein assay kit (Bio-Rad). Equal amounts of proteins were prepared in protein lysis buffer containing a final concentration of 15% glycerol, 50mM DTT and bromophenol blue. The samples were boiled at 95°C for 5 min and resolved by western blotting. Membranes were probed with the following antibodies: anti-Ripk3 (Cell signaling #95702), anti-CHD4 (Cell signaling #12011) and anti-GAPDH (Cell Signaling #2118). Quantification was performed by densitometric analysis using Image Lab 5.0 software (Bio-Rad).