Fig. 4. P63RhoGEF induces RhoA-mediated stress fiber formation. (A) REF-52 cells were transfected with the different constructs as indicated and, 15 hours later, cells were fixed and permeabilized as described in Materials and Methods. The expression of p63RhoGEF was detected by an anti-His monoclonal antibody followed by FITC-coupled anti-mouse immunoglobulins, whereas F-actin was detected by rhodamine-conjugated phalloidin. Expression of GFP-RhoAV14 was visualized directly. When indicated, purified C3 exoenzyme was added in cell medium (0.5 µg/ml) 15 hours before fixation. This experiment is representative of at least three independent experiments, and 100 cells were examined each time. Transfected cells are indicated by white arrows. (B) Quantification of enhancement of stress fiber formation in REF-52 cells transfected as indicated.