FIGURE 6.

UBE2H is required for MG53-induced FAK ubiquitination. A and B, C2C12 myoblasts were treated with si-control (si-con) or si-UBE2H (100 nm) for 24 h and then differentiated into myotubes for 3 days. The expression levels of FAK, UBE2H, MyHC, Cav-3, and GAPDH were determined by immunoblotting (A). The expression of FAK was also quantified by densitometry. These experiments were repeated three times (Student's t test; *, p < 0.01) (B). C, UBE2H knockdown C2C12 myotubes were treated with MG132 (5 μm) for 12 h, and FAK ubiquitination was determined by endogenous immunoprecipitation (IP). WCL, whole cell lysate. D, UBE2H overexpression increases MG53-induced FAK ubiquitination. HEK 293 cells were cotransfected with different combinations of FLAG-FAK (1.5 μg), His-Ub (0.5 μg), HA-MG53 (0.5 μg), and Myc-UBE2H (0.5 μg) for 24 h. After MG132 treatment for 12 h, FAK ubiquitination was determined by immunoprecipitation with an anti-FLAG antibody and immunoblotting with an anti-His antibody.