RPE cells grown to 80% confluence were serum-starved overnight. EphrinB2/Fc, EphB4/Fc (both from R&D Systems) or Fc fragment (Jackson Laboratories, Bar Harbor, ME) alone was clustered by incubation with anti-human Fc antibody (1:1000 dilution; Jackson Laboratories) for 1 hour at 4°C and were added to the culture medium at a concentration of 2 μg/mL, with or without sEphB4 (3 μg/mL) for 20 minutes. To study the role of PDGF, serum-starved cells were stimulated with PDGF (10 ng/mL) for 20 minutes. After stimulation, media were aspirated and cells were immediately harvested with protein extraction buffer containing 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% (vol/vol) Triton X-100, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 1 mM sodium vanadate, pH 7.8. Protein extracts were clarified by centrifugation at 18,000g for 20 minutes at 4°C. Cleared cell lysates were incubated with 2 μg/mL EphB4 monoclonal antibody (clone 47; Vasgene Therapeutics, Inc.) or EphrinB2 monoclonal antibody (clone 2B5; Vasgene Therapeutics, Inc.) for 2 hours. Antigen-antibody complexes were immunoprecipitated by shaking with 10 μL protein G-Sepharose beads (Santa Cruz Biotechnology, Inc.) for 1 hour at 4°C. Immunoprecipitates were immunoblotted with anti-phosphotyrosine antibody (clone 4G10; Upstate Biotechnology, Lake Placid, NY) to detect phosphorylation status. To monitor immunoprecipitation efficiency, a duplicate membrane was probed with EphB4-specific (clone 265; Vasgene Therapeutics, Inc.) or EphrinB2-specific (clone P20; Santa Cruz Biotechnology) antibody.