Detection of EphB2 and EphB4 phosphorylation in primary human endothelial cells. (A) Kinetics of EphB2 phosphorylation in HUVECs stimulated with ephrin B1-Fc. HUVECs were incubated in medium only or were treated with ephrin B1-Fc (1 μg/mL) for 10 minutes, 30 minutes, 2 hours, and 4 hours. Cell lysates (150 μg) were immunoprecipitated with goat IgG antibodies to EphB2, and the precipitates were immunoblotted with anti–phosphotyrosine mouse monoclonal antibodies (4G10). The membrane was reblotted with goat IgG antibodies to EphB2 (results are representative of 3 experiments). (B) Effect of the SNEW peptide (100 μM) on EphB2 phosphorylation in HUVECs stimulated for 10 minutes with ephrin B1-Fc (1 μg/mL) or with a control Fc-fusion protein (Fc, leptin receptor-Fc chimera, 1 μg/mL). As a control peptide (control), we used a scrambled form of SNEW (SCR-EPQ, 100 μM). EphB2 was immunoprecipitated, immunoblotted with anti–phosphotyrosine mouse monoclonal antibodies, and reblotted with goat IgG antibodies to EphB2 antibodies. (C) EphB4 phosphorylation was induced in HUVECs stimulated with ephrin B2-Fc (1 μg/mL). HUVECs were incubated for 30 minutes with a control Fc-fusion protein (Fc, leptin receptor-Fc chimera, 1 μg/mL) or with ephrin B2-Fc (1 μg/mL) in medium alone or in medium supplemented with the peptide TNYL-RAW (100 μM) or the peptide SNEW (100 μM). EphB4 was immunoprecipitated, immunoblotted with anti–phosphotyrosine mouse monoclonal antibodies, and reblotted with goat IgG antibodies to EphB4 antibodies. (D) EphB2 phosphorylation was induced in HUVECs after stimulation for 30 minutes with ephrin B2-Fc (1 μg/mL) but not with a control Fc-fusion protein (Fc, leptin receptor-Fc chimera, 1 μg/mL). Phosphorylated EphB2 was visualized after immunoprecipitation and immunoblotting with anti–phosphotyrosine mouse monoclonal antibodies; total EphB2 was visualized after reprobing with antibodies to EphB2.