Figure 5.

Inhibition of Rho signaling in GEβ1 cells. (A) Rac and Rho activity assay in GEβ1 and GEβ3 cells. (B) GEβ1 or GEβ3 cells transiently transfected with the indicated expression plasmids in combination with GFP cDNA as a transfection marker (insets) were seeded on FN-coated coverslips 24 h after transfection for 12 h and were fixed, permeabilized, and stained for F-actin. Arrows indicate transfected cells. Bars, 10 μm. (C) Analysis of GFP-paxillin dynamics in cell–matrix adhesions of GEβ1, GEβ3, and Y27632-treated GEβ1 cells. Shown is the halftime of loss of fluorescence (τ) ± SEM calculated from FLIP experiments such as depicted in Fig. 4 D. (D) Control or C3-transfected GEβ1 cells (indicated by GFP; inset and arrow) were plated overnight on FN-coated coverslips and stimulated with PMA for 1 h in the absence or presence of Y27632 as indicated. Preparations were fixed, permeabilized, and stained for F-actin. Filled arrowheads indicate Y27632-induced membrane ruffles/lamellipodia. Bars, 5 μm. (E) GEβ1 cells were plated overnight on FN-coated coverslips, confluent monolayers were wounded with a micropipette tip, and preparations were fixed, permeabilized, and stained for F-actin after 5 h incubation in the absence or presence of Y27632. Open arrowheads indicate the direction of the wound; filled arrowheads indicate Y27632-induced protrusions of cells moving into the wounded area. Bar, 10 μm. (F) GEβ1 cells were plated on FN-coated coverslips for the indicated times in the absence or presence of Y27632 as indicated. Western blot analysis of total lysates with the indicated antibodies is shown. (G) Mean ± SD of relative cofilin Ser3 phosphorylation determined from two individual experiments such as depicted in F.