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Fig. 3. Screening of a protease that cleaves the extracellular domain of ephrin-B1. (A) Panc-1 cells transiently expressing Flag-tagged ephrin-B1 were incubated with various protease inhibitors for 4 hours in medium containing 0.5% FBS: leupeptin and E64d (Loxistatin), a cystein protease inhibitor; GM6001, a pan-MMP inhibitor; PD150606, a calpain inhibitor; pepstatin A, an aspartic protease inhibitor; DCI (3,4-dichloroisocoumarin), a serine protease inhibitor; TPCK (N
-tosyl-phe chloromethyl ketone), a chymotrypsin inhibitor. The cleavage of ephrin-B1 ectodomain was examined as described in Fig. 2A. (B) Left: SUIT-4 cells were incubated with EphB2-Fc (2 µg/ml) together with or without TIMPs (100 nM for each) as indicated for 2 hours. The processed ephrin-B1 fragment was detected in the culture medium. Right: THP-1 cells were treated with PMA (10 ng/ml) together with or without TIMP-3 (100 nM) for 6 hours. Open arrowhead indicates the processed fragment of TNF- in the medium detected by immunoprecipitation with anti TNF- antibody. (C) SUIT-4 cells were treated with various MMP inhibitors as indicated (1 µM for MMP-8 inhibitor and 5 µM for others) for 4 hours. Ephrin-B1 fragment was detected in the medium. (D) Purified ephrin-B1-Fc protein was incubated with activated MMP in vitro for 1 hour at 37°C, separated by SDS-PAGE, and immunoblotted with anti-Fc mouse IgG or anti-ephrin-B1. Bottom: a schematic representation of ephrin-B1-Fc with the MMP-8 cleavage site indicated by a dotted line. Open and filled arrowheads indicate uncleaved ephrin-B1-Fc and the processed fragments, respectively.
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