Figure 6

Expression of a drebrin mutant insensitive to BTP2 rescues myotube formation in BTP2-treated C2C12 cells. (A) C2C12 cells were transfected with vectors encoding GFP, drebrin E-GFP or drebrin E (K270M, K271M)-GFP. Transfection efficiency was measured as > 70% by visualization of GFP. Forty-eight hours after transfection, expression of exogenous and endogenous drebrin was examined by Western blot analysis with antibodies to GFP or drebrin (Dbn). (B) Forty-eight hours after transfection, cells were switched to differentiation medium with or without 5 μM BTP2 as indicated. Seventy-two hours later, cultures were fixed and immunostained for myosin heavy chain (MHC; red). GFP and exogenous drebrin expression were visualized by direct fluorescence (green). Nuclei were counterstained with 4',6-diamidino-2-phenylindole (blue). KM, KM-GFP; drebrin E (K270M, K271M)-GFP. (C) Quantification of the fusion index in cultures from (A). Values shown are means ± SD of triplicate determinations. *P < 0.01 as compared to BTP2-treated GFP-expressing cultures.