Analysis of EphB2 and EphB4 phosphorylation and function during endothelial cell cord formation on extracellular matrix. (A-B) HUVECs were incubated on Matrigel-coated wells for the indicated times. After incubation, cells were washed in PBS at 4°C and removed by trypsin digestion at 4°C, and cell lysates were immunoprecipitated with goat IgG antibodies to EphB2 (A) or EphB4 (B). Immunoprecipitates were immunoblotted with anti–phosphotyrosine mouse monoclonal antibody 4G10, and the membranes were reblotted with the immunoprecipitating antibodies to EphB2 or EphB4. Results derived from independent experiments, each representative of 3 experiments. (C) Effects of the peptides CTCE-9908, SNEW, and TNYL-RAW on Matrigel-dependent cord formation. HUVECs were incubated for 18 hours onto Matrigel-coated wells in medium alone (none), with control peptide SCR-WTL (100 μM), CTCE-9908 peptide (50 μg/mL), SNEW peptide (100 μM), or TNYL-RAW peptide (100 μM). Representative images from phase-contrast microscopy reflecting various degrees of cord formation after 18 hours of incubation (original magnification, ×5). Representative results from 5 experiments. (D) Quantitative analysis of HUVEC cord formation on Matrigel-coated wells under the same conditions listed in panel C and measured as a function of the number of vascular joints per visual field (magnification, ×10). Results reflect the mean ± SD of 3 independent experiments; in each experiment, 4 nonoverlapping fields were counted.