Fig. 4. MMP-8 is the key protease of ephrin-B1 cleavage. (A) Expression of MMP-8 in cell lysates. Left: The indicated cells were seeded on plates not to reach confluence. Cell lysates were prepared on the day after plating. Right: cell lysates were prepared 4 hours (4h) or 3 days (d3) after being plated on dishes. The cells were confluent on day 3 after plating. (B) SUIT-4 cells treated with either MMP-8 siRNA or control scrambled siRNA (control), or left untreated. The cells were detached 48 hours later, replated on new plates and further incubated for 24 hours. Left: Cellular levels of MMP-8 were analyzed 72 hours after treatment of SUIT-4 cells with siRNAs. Right: the culture medium was replaced with one fresh medium or medium containing EphB2-Fc (2 g/ml) and incubated for 2 hours to detect ephrin-B1 ectodomain in the medium. (C) SUIT-4 cells were transiently transfected with the indicated volume of a plasmid encoding Flag-tagged activated MMP-8 cDNA. After 48 hours of transfection, the medium was replaced and the cells were further incubated for 6 hours to detect processed ephrin-B1 ectodomain in the medium. The expression of transfected MMP-8 in each cell lysate was confirmed by immunoblotting with anti-Flag antibody (bottom). (D) The lysate of L ephrin-B1 cells was immunoprecipitated with anti-MMP-8 polyclonal antibody or control rabbit IgG1, and subjected to immunoblotting with anti-ephrin-B1 C18 antibody. HRP-conjugated anti-rabbit IgG (TrueBlot) was used as the secondary antibody for immunoblotting to avoid cross reaction with denatured rabbit IgG heavy chain of the antibody used for immunoprecipitation. The arrowhead indicates coprecipitated ephrin-B1. The asterisk indicates the IgG light chain.