Expression and characterization of sEphB4 and sEphB4-HSA. (A) sEphB4-HSA was expressed in CHO cells and purified to near homogeneity and separated on SDS-PAGE (Coomassie staining) under reducing and nonreducing conditions. (B) Saturation binding kinetics of sEphB4 and sEphB4-HSA in a solid-phase ELISA. Interaction of increasing concentrations of sEphB4 or sEphB4-HSA with sEphrinB2-AP was determined in a solid-phase ELISA. Each point was determined in triplicate. Dissociation constants were calculated with the use of nonlinear regression and Graphpad Prism. (C) Systemic pharmacokinetics of sEphB4 and sEphB4-HSA administered intraperitoneally. Mice were injected with a 10 mg/kg dose of either sEphB4 or sEphB4-HSA administered intraperitoneally. Each point represents the average of 2 separate experiments. Error bars represent the SEM. (D) Pharmacokinetic and saturation binding constants of sEphB4 and sEphB4-HSA. (E) Tyrosine phosphorylation of EphB4 receptor in MCF7 cells in response to stimulation with EphrinB2-Fc (15 minutes) in the absence or presence of EphB4-derived soluble proteins.