Fig. 6. Effect of EphB4-Fc and ephrinB2-Fc on sprouting angiogenesis of mock and EphB4 (clone 4-8)-transfected PAECs. (A) PAEC spheroids were embedded in collagen gels and stimulated with ephrinB2-Fc or EphB4-Fc for 48 hours. PAECs have a high baseline sprouting that was set to 100%. The sprouting activity of EphB4-transfected PAECs was significantly inhibited upon treatment with ephrinB2-Fc (***P<0.001) but not upon EphB4-Fc treatment. By contrast, mock-transfected PAECs do not respond to either ephrinB2-Fc or EphB4-Fc treatment. (B) EphB4-overexpressing PAECs were stimulated with ephrinB2-Fc for 30 minutes and analyzed for EphB4 phosphorylation by immunoprecipitating EphB4 using ephrinB2-Fc. Blots were probed with an anti-EphB4 antibody (left) and reprobed with an anti-pTyr antibody (right). EphrinB2-Fc stimulation leads to prominent phosphorylation of EphB4 in PAECs overexpressing mEphB4.