Fig. 3. Interrelation of Rac and Rho activity in Tiam1-expressing melanoma cells. (A) GTPase pull-down assay of parental cells (1F6, MV3) and Tiam1-expressing cells (1F6-Tiam1, MV3-Tiam1). Cell lysates of the indicated cell lines (3x10⁶ cells for Rac assay, 6x10⁶ cells for Rho assay) were incubated with GST-PAK-CD (upper panel) or GST-C21 (lower panel), and bound GTPases were detected by western blot using antibodies against Rac1 and RhoA. (B) Rac and Rho activities were visualized indirectly by staining F-actin (red). Focal adhesion complexes were visualized using anti-vinculin antibody (white). Tiam1 was stained with pAb and FITC-conjugated secondary antibody (green). Note that increased Rac activation did not lead to reduction of stress fiber or focal adhesion formation, indicating a stable level of Rho activity and that Tiam1 colocalizes with F-actin at the cell-cell contact sites in MV3-Tiam1 cells. Bars, 20 µm.