Figure 6

Scambio stimulates guanine nucleotide exchange on RhoA and RhoC Cells were transfected with RhoA and vector (lanes 1, 2), with RhoA and Dbl (lanes 3, 4), with RhoC and vector (lanes 5, 6), with RhoC and Scambio (lanes 7, 8) or with RhoA and Scambio (lanes 9, 10). Cells were stimulated with either 10% fetal calf serum for one minute (indicated as + below the panel) or were further serum-starved immediately prior to lysis (indicated as -). The amount of GTP-Rho and total Rho was quantitated by immunoblotting with anti-Xpress antibodies on the pull-down (upper, GTP-Rho) or 1.7% of the total cellular lysate (middle, total Rho). Equal application of GST-RBD in the Rhotekin pull down assay is shown by staining of the gel with Coomassie Brilliant Blue (bottom, GST-RBD). The fold activation was determined by normalizing the GTP-Rho levels to total Rho levels and was measured relative to the level of activation seen for the control, using densitometric quantification. Similar results were obtained in more than two other independent experiments.