Fig. 5. Co-transfection of Rac1N17, but not of RhoN19 or Cdc42N17, abrogates gelsolin-induced collagen type I invasion of HEK293T cells. (A) Epifluorescence images of HEK293T cells transiently transfected with G-GFP (upper left) or EGFP (lower left). Right panels show corresponding DAPI staining of nuclei. Bar = 50 µm. (B) Collagen invasion. Upper panel: invasion index levels (mean ± SE, n = 3) are shown for parental HEK293T cells (lane 1, negative control) and gelsolin-transfected HEK293T cells (293G-GFP, lane 2). Lanes 4–9 represent co-transfections between G-GFP and activated or inactivated Rac1 (lanes 4 and 5), RhoA (lanes 6 and 7) or Cdc42 (lanes 8 and 9). Lanes 3 and 10–13 are controls. Lane 14, fragmin does not induce invasion. EV, empty vector. Middle panel: western blot on cytosolic proteins from transfected HEK293T cells showing equal expression levels of G-GFP or tubulin. The numbers at the top correspond to numbers in the histogram. Lower panel: EGFP expression (upper autoradioagram) in cells that were double transfected with empty vector or activated GTPases. Lower autoradiogram: tubulin. Fragmin was visualized using affinity-purified anti-fragmin antibodies. (C) Western blots showing expression of Rac, Rho or Cdc42, co-transfected with G-GFP (left panel) or with EGFP (right panel). DN, dominant-negative; CA, constitutively active.