Fig. 1. EphB1/Fc stimulates endothelial ephrin-B1 tyrosine phosphorylation. Upper panel, ephrin-B1 is expressed in human renal microvascular endothelial cells (HRMEC). HRMEC or CHP100 (Davis et al., 1994) were surface biotinylated as described in the Materials and Methods, and ephrins were immunoprecipitated using non-immune rabbit serum (NRS) or rabbit polyclonal anti-ephrin-B1 antibodies recognizing C-terminal sequences (P1) or anti-ephrin-B1 recognizing an ephrin-B1-specific juxtamembrane spacer domain peptide (P2). Immunoprecipitated complexes were separated on a 10% SDS-PAGE under non-reducing conditions, transferred to PVDF membranes and detected with streptavidin-HRP using enhanced chemiluminescence (Amersham). The ephrin-B1-specific antibody (P2) was used in all subsequent experiments. Lower panel, phorbol myristate acetate and EphB1/Fc stimulate ephrin-B1 tyrosine phosphorylation. Serum-depleted HRMEC were replated on fibronectin-coated p60 tissue culture dishes for 60 minutes, then stimulated for 15 minutes at 37°C with vehicle (NA), phorbol myristate acetate (PMA, 20 ng/ml), control IgG1 (2 µg/ml) or EphB1/Fc at the indicated concentrations. Cells were lysed in RIPA buffer, immunoprecipitated with rabbit anti-ephrin-B1, and the levels of ephrin-B1 tyrosine phosphorylation were determined using the monoclonal antibody 4G10 conjugated to HRP followed by ECL detection. The results are representative of five independent experiments.