Fig. 5. Stimulation of ephrin-B1 with EphB2 increases MMP-8 secretion. (A) SUIT-4 cells were left untreated or were treated with EphB2-Fc or co-cultured with either parent or EphB2-expressing HEK293 cells for 4 hours. Cellular levels of MMP-8 were analyzed by RT-PCR using GAPDH as a control. (B) SUIT-4 cells were incubated with or without EphB2-Fc for 2 hours in the presence or absence of cyclohexamide (CHX, 100 µg/ml) or actinomycin D (ACND, 5 µg/ml) to detect the ephrin-B1 ectodomain in the medium. (C) SUIT-4 cells or PANC-1 cells stably expressing ephrin-B1 were treated with actinomycin D (5 µg/ml) together with or without EphB2-Fc (2 µg/ml) for 4 hours. Cell lysates were prepared and intracellular expression levels of MMP-8 were analyzed by western blotting using -tubulin as a loading control. (D) Conditioned medium of SUIT-4 cells were collected after the cells were treated with EphB2-Fc or left untreated for 4 hours in serum-free medium. Proteins secreted in the medium were precipitated with trichloroacetic acid (10%), resuspended in sample buffer, and subjected to immunoblotting with anti MMP-8 polyclonal antibody. Arrowheads indicate MMP-8 protein (proenzyme and activated). (E) SUIT-4 cells were metabolically labeled with [³⁵S]methionine, then treated with EphB2-Fc or control Fc for 2 hours. The amount of labeled MMP-8 (left panel) or MMP-7 (right panel) in the medium was evaluated through immunoprecipitation from the conditioned medium followed by SDS-PAGE and autoradiography. Arrowhead indicates MMP-8 (proenzyme and activated; left) or MMP-7 (right) in the medium. (F) Wild-type and mutants of ephrin-B1 were expressed in Capan-1 cells as in Fig. 2A, and [³⁵S]methionine-labeled MMP-8 was detected in the medium (left panel). Right: The total amount of MMP-8 in the conditioned medium of Capan-1 cells was evaluated using the trichloroacetic acid (TCA) precipitation procedure, followed by immunoblotting with anti MMP-8 antibody as in D.