Figure 3

Effect of plexinB1 mutation on RhoA activation. A). Mutation of plexinB1 does not affect PDZRhoGEF (PRG) binding. Lysates of HEK293 cells co-expressing VSV-plexinB1 and PDZRhoGEF were immunoprecipitated with anti-VSV and bound proteins detected by western blotting. B). Mutation of plexinB1 does not affect RhoA activation. Lysates of HEK293 cells co-expressing VSV-plexinB1, FLAG-PDZRhoGEF and myc-RhoA were immunoprecipitated with GST-Rhotekin and RhoGTP detected by western blotting with anti-myc. C). COS-7 cells seeded onto coverslips were either transfected with plexinB1 (iii and iv) or vector (i and ii). Fixed cells were stained with anti-plexinB1 (ii and iv) or for actin (i and iii). Panel i and ii, and iii and iv, show the same field. Scale bar = 20 μm. D). The number of cells possessing more than three straight actin bundles of at least 5 μm long was counted and expressed as a percentage of the total number of cells in the field. Bars represent means +/- SE. D). Sema4D in conditioned medium. Conditioned medium was collected from Cos7 cells transfected with vector or Sema4D-Fc and the protein detected by immunoblotting with anti-Sema4D antibody.