FIGURE 5.

SUMO-modified Sharp-1 inhibits MyoD transcriptional activity and function. A, 10T1/2 cells transfected with MyoD, Myc-Sharp-1, and Myc-Sharp-1 2KR were analyzed for expression of MyoD and Sharp-1 by Western blotting. B–D, myogenic conversion assays were performed in cells transfected with MyoD, MyoD, and Sharp-1, or MyoD and Sharp-1 2KR. 6 days later, differentiated cells were stained with anti-MHC antibody (red). Nuclei were stained with DAPI (B). Differentiation was quantified by plotting myogenic index (C). Troponin T expression was assessed by Western blotting (D). E, 10T1/2 cells were transfected with pMyog-Luc promoter (100 ng) together with MyoD (50 ng), Sharp-1 (25 and 50 ng), or Sharp-1 2KR (50 ng) as indicated. 48 h later, cells were harvested and assayed for luciferase activity. Error bars indicate mean ± S.D. F, C2C12 cells were transfected with Myc-tagged Sharp-1 and Sharp-1 2KR. 24 h later, lysates were immunoprecipitated, and interaction of Sharp-1 and Sharp-1 2KR with endogenous MyoD was analyzed by Western blotting with anti-MyoD antibody. Lysates (input) were immunoblotted with anti-Myc and anti-MyoD antibodies to detect Sharp-1and MyoD expression.