Fig. 4. A dominant negative form of p120RasGAP prevents the decrease in R-RasGTP levels and COS cell retraction induced by ephrins. (A,B) A COS cell line stably transfected with R-Ras was transiently transfected with a dominant negative form of p120RasGAP (GAP-N) or pcDNA3 vector control and stimulated with ephrin-A1 Fc, ephrin-B1 Fc or Fc as a control. GTP-bound R-Ras was isolated with GST-Raf1 RBD and detected with anti-R-Ras antibodies in duplicate samples for the GAP-N transfected cells. (C) The histogram shows the relative levels of R-Ras GTP quantified from the experiment in A and B and normalized to the R-Ras levels in the lysates. (D) COS cells were transiently transfected with EGFP-tagged GAP-N or EGFP vector as a control. Cells were stimulated with ephrin-A1 Fc, ephrin-B1 Fc or Fc as a control and stained with rhodamine-phalloidin (red). Bar, 20 µm. (E) Histogram showing the mean percentage of cells that have spikes at the periphery; bars represent standard errors from three experiments. GAP-N-transfected cells treated with ephrin-A1 Fc or ephrin-B1 Fc were compared with similarly treated control-transfected cells by one-way ANOVA and Tukey's post-hoc test, ^**P<0.01 and ^***P<0.001.