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Figure 2


Fig. 2. Activation of ephrin-B1 cleavage requires its C-terminus. (A) Wild-type and various mutants of ephrin-B1 tagged with Flag at the N-terminus were expressed in Capan-1 cells by retrovirus-mediated gene transfer. The cells were treated with EphB2-Fc (2 µg/ml) for 1.5 hours (+) or left untreated (–) and conditioned medium was assayed for the presence of ephrin-B1 fragments by immunoprecipitation (IP) and immunoblotting (IB) with anti-flag antibody. The filled arrowhead indicates the ephrin-B1 ectodomain fragment. (Bottom) Expression of wild-type or mutated ephrin-B1 in cell lysates after treatment with EphB2-Fc. The open arrowhead indicates ephrin-B1 {Delta}C19. (B) SUIT-4 cells were treated with EphB2-Fc and PP2 or control PP3or left untreated, as indicated. Conditioned medium was collected after 2 hours and subjected to immunoprecipitation and immunoblotting. In the bottom panel, suppression of tyrosine phosphorylation of ephrin-B1 by PP2 treatment is shown. # and * indicate the IgG heavy chain and light chain, respectively.