Fig. 5. EphA2-mediated Rac1 activation and migration is dependent on PI3K. (A) Active GTP-bound form of Rac1 was analyzed by Pak-PBD pull-down assay using lysates from ephrin-A1-stimulated BPMEC in the presence or absence of PI3K inhibitors, wortmannin or LY294002. Total Rac1 levels within the lysate prior to PBD-pulldown were detected by immunoblot. Data are representative of three independent experiments. (B) Migration of ephrin-A1-stimulated BPMEC, in the presence or absence of LY294002 PI3K inhibitor, was 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 vehicle + ephrin-A1 versus LY294002 + ephrin-A1. (C) Activation of Rac1 was determined by Pak-PBD pull-down assay in BPMEC expressing a dominant negative p85 PI3K regulatory subunit (-p85) in response to ephrin-A1 stimulation. (D) Migration of BPMEC transfected with -p85 in response to ephrin-A1 was 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 -p85 + ephrin-A1. Expression of -p85 was confirmed by immunoprecipitation of p85 (endogenous and truncated -p85) followed by p85 immunoblot.