Structure and biochemical properties. (A) Schematic illustration of truncated soluble proteins. (B) SDS-PAGE. Coomassie staining of EphB4-derived soluble proteins. (C) Binding of EphrinB2-AP fusion protein to EphB4 soluble proteins immobilized on Ni-NTA agarose beads. Results of 2 independent experiments are shown for each protein. Experiments were repeated 3 times (D) Tyrosine phosphorylation of EphB4 receptor in HUVECs in response to stimulation with EphrinB2-Fc (15 minutes) in the absence or presence of EphB4-derived soluble proteins. (E) Tyrosine phosphorylation of EphrinB2 by EphB4 in the presence of sEphB4. 293T cells transiently transfected with full-length EphrinB2 and EphB4 expression vector were cocultured (15 minutes) with or without sEphB4 for 24 hours (see “Monomeric sEphB4 antagonizes forward and reverse signaling” for details). Total amount of EphrinB2 by Western blot (bottom panel) and phosphorylation status of EphrinB2 after immunoprecipitation (IP) (top panel) are shown. Experiments were repeated twice.