FIGURE 6.

Time-dependent effect of FKHR on MRTF-A-induced activation of the Id3 promoter during myogenic differentiation. A, myoblasts (GM) and differentiated myocytes (DM) cultured for 4 days in DM were separated into the cytoplasmic (C) and nuclear (N) fractions, followed by immunoblotting for MRTF-A. Immunoblotting for HSP-90 and histone H2B indicates control for a cytoplasmic protein and a nuclear protein, respectively. B, cells (cultured in GM and GM containing 25 μm of Y-27632 and 4day cultures in DM) were stained using the anti-FKHR antibody. Magnification is ×600. C, proliferating C2C12 myoblasts were transfected with Id3 (-2000/+55)-Luc, pSV-β-gal, ca-MRTF-A expression plasmid (80 (+) or 160 ng (++)) and increasing amounts of FKHR expression plasmid (0, 80, 160, or 320 ng). The transfected plasmids were adjusted to a constant amount by the addition of empty plasmid. Luciferase activity was measured at the proliferation (24 h after transfection in GM) and differentiation (2 days in DM) phases. The luciferase activities were expressed relative to that in empty plasmid-transfected C2C12 cells, which was set as 1.0.