Fig. 4. EphA2-mediated endothelial cell migration is regulated by Rac1 GTPase. (A) Active GTP-bound forms of Rac1 and cdc42 were analyzed by Pak-PBD pulldown followed by immunoblot in lysates from heterozygous (+/–) or EphA2-deficient (–/–) MPMEC stimulated with ephrin-A1. Total Rac1 and cdc42 levels within the lysate prior to PBD-pulldown were detected by immunoblot. Data are representative of four independent experiments. (B) Activation of Rac1 induced by ephrin-A1 stimulation was confirmed in BPMEC, and occurred upon initiation of EphA2 autophosphorylation. (C) Ephrin-A1-induced migration of mock transfected or EphA2-Neu^TM-expressing BPMEC in the presence or absence of GTPase inhibitor Toxin B was quantified by transwell assay. Significant differences in migration are indicated where P<0.01 using Student's t-test: ^*P=0.0001 mock + ephrin-A1 versus mock + ephrin-A1/Toxin B, ^**P=0.0005 EphA2-Neu^TM versus EphA2-Neu^TM/Toxin B. (D) Migration of BPMEC transfected with an expression construct encoding dominant negative Rac1 (Rac1-N17) in response to ephrin-A1 was also quantified by transwell assay by scoring three fields per transwell for each condition in triplicate samples. Data are means±s.d. of three independent experiments. Significant differences in migration are indicated where P<0.01 using Student's t-test: ^***P=0.0005 mock + ephrin-A1 versus Rac1-N17 + ephrin-A1. Expression of Rac1-N17 was confirmed by myc immunoblot.