Inhibition of constitutive and inducible JAM-A shedding. (A) HEK293 cells and HUVECs were incubated with the preferential ADAM10 inhibitor GI254023X (GI, 5 μM), the combined ADAM10/17 inhibitor GW280264 (GW, 5 μM), or vehicle control (DMSO) for 16 hours. Conditioned media were analyzed by Western blotting using an anti–hJAM-A monoclonal antibody. (B) Surface expression of JAM-A on HEK293 cells treated with DMSO or inhibitor as described in panel A was analyzed by flow cytometry. (C) Cells were pretreated with the ADAM10 inhibitor GI254023X (5 μM) or ADAM10/17 inhibitor GW280264X (5 μM) or DMSO. Subsequently, cells were incubated in the presence or absence of PMA (200 ng/mL) for 2 hours. Supernatants were harvested, and soluble JAM-A was determined by Western blotting using an anti–hJAM-A monoclonal antibody. (D,E) Cells were stimulated with TNF-α/IFN-γ (10 ng/mL) or PAF (50 nM) or treated with vehicle control (DMSO) for 16 hours in the absence or presence of GI254023X or GW280264 (both 5 μM), and released JAM-A was determined by Western blotting. Quantified signals in panels A through E were normalized and shown as mean plus or minus SD for 3 independent experiments. *Statistically significant reduction in JAM-A surface expression caused by cell stimulation (P < .05).