Fig. 7. MyoD DRR activity is inhibited by mDia. (A) Gel-shift assays of the putative TCF/LEF sites in the MyoD DRR. ³²P-end-labeled oligo probes representing a TCF consensus site or two of the three sites from the MyoD DRR (denoted DRR site A or DRR site B) were incubated with extracts prepared from control C2C12 myoblasts (indicated as `c') or myoblasts treated with 2.5 µM BIO for 24 hours. Assays were performed in the presence or absence of 100-fold molar excess of the respective cold competitor oligo. The consensus TCF site participated in the formation of complexes that were competed by excess cold probe, and induced by BIO (black arrow) consistent with the binding of -catenin–TCF. The gray arrow indicates a nonspecific complex. However, DRR sites A and B bound a nonspecific complex (indicated by ^*) that was neither competed nor BIO-inducible. Thus, the TCF consensus sites in the MyoD DRR do not appear to function as targets of specific nuclear factor binding. (B) MyoD DRR activity is inhibited by mDia. Myoblasts were transiently transfected in growth medium with a mouse MyoD DRR-pGL3 promoter construct along with pBS (control) or mDia constructs, and a gal plasmid. Luciferase activity was quantified after 24 hours and normalized for transfection efficiency (mean ± s.e.m., n=3). N3 is the most effective at suppressing DRR activity, and overall the DRR suppressive activity of the different forms of mDia1 correlated with suppression of TCF activity.