Fig. 1. The ectodomain of ephrin-B1 is secreted into the culture medium of human pancreas cancer cells. (A) Various pancreas cancer cell lines were cultured in medium containing 0.5% FBS. After 6 hours, conditioned medium was collected and subjected to immunoprecipitation (IP) and immunoblotting (IB) with polyclonal antibodies against the extracellular domain of ephrin-B1 (ephrin-B1 ECD). The immunoprecipitated 35 kDa ephrin-B1 fragment is indicated by an arrowhead. The expression of ephrin-B1 in each cell lysate was confirmed by immunoblotting (bottom). (B) A diagram of ephrin-B1 is shown at the top. TM, transmembrane domain; Y, tyrosine phosphorylation sites. Dotted line indicates the cleavage site of ephrin-B1, and the PDZ domain-binding motif is indicated as a gray box at the C-terminus. Four aa residues around the cleavage site were changed to alanine (ephrin-B1 4A) destroying the MMP-8 cleavage site. The conditioned medium of COS-1 cells transfected with wild-type (wt) or mutant ephrin-B1 (4A), or independent PANC-1 clones stably expressing ephrin-B1 were collected and subjected to immunoprecipitation and immunoblotting as described in A. (C) SUIT-4 cells were either treated with EphB2-Fc (4 µg/ml) for 2 hours (+) or left untreated (–). The ephrin-B1 fragment in the medium was detected as in A (left) or the cell lysates were subjected to immunoblotting with anti ephrin-B1 C18 (right panel). Open and filled arrowheads indicate uncleaved ephrin-B1 and its processed fragment (p17), respectively. (D) SUIT-4 cells were transiently transfected with ephrin-B1 tagged with HA at the C-terminus. Transfected SUIT-4 cells were overlayed on a monolayer of parent HEK293T cells or HEK293T cells stably expressing EphB2 for 2 hours. Cell lysates were prepared from co-cultured cells to detect HA-tagged p17 fragment derived from exogenously expressed ephrin-B1 in SUIT-4 cells by immunoprecipitation. # and ^* indicate the IgG heavy chain and light chain, respectively. Open and filled arrowheads indicate uncleaved ephrin-B1 and its processed fragment (p17), respectively.