Figure 3.

Transient expression of activated Rac1 and Cdc42 alleles, but not of RhoA, is sufficient to inhibit the expression of myogenin and myosin in QMb. Primary QMb (A) and QMb-LA29 (B) at 35°C were transiently transfected with expression vectors for β-galactosidase (β-Gal) and myc epitope–tagged Rac1V12, Rac1L61, Rac1L61A37, Rac1L61C40, Cdc42L61, RhoAV14, and Rac1N17. After transfection, QMb and QMb-LA29 were cultured in DM containing 1.85 mM EGTA for 2 d at 37 and 41°C, respectively. After fixation, cells were double labeled with mAbs to β-galactosidase or to the myc tag and with polyclonal antibodies to chicken myogenin, a muscle-specific regulatory factor, or to chicken myosin. Double scoring of cells for either β-Gal/myogenin and β-Gal/myosin or myc/myogenin and myc/myosin is shown as percentage of cells expressing the transfected proteins. Very similar results were obtained when β-Gal was substituted with GFP. Transfection followed by immunolocalization was performed in five independent experiments, and in all experiments at least 200 transfected cells were examined for each condition.