FIGURE 5.

Effect of specific inhibitors of the ADAM family of proteases on meprin β shedding in meprin β and meprin αβ clones. A, HEK293 cells stably transfected with meprin β were treated with highly specific inhibitors of ADAM10 and ADAM17 in the presence or absence of PMA (30 ng/ml) for 4 h. The inhibitors used were GI254023X (2 μm) and GW280264X (2 μm). Medium supernatants were centrifuged for 30 min at 100,000 × g to remove cellular debris, and aliquots of equal protein content were analyzed by Western blot for the presence of meprin β (left panel). The individual lanes as shown were obtained from the same Western blot. Recombinant human promeprin β lacking its membrane anchor purified from HEK293 cells described earlier (21) is shown as a reference. Western band intensities were evaluated by densitometry normalized to those observed in medium supernatant of the corresponding untreated clones (right panel). Error bars represent S.E., n = 3. **, p < 0.01, ***, p < 0.001. B, HEK293 cells stably co-transfected with meprin αβ (meprin A) were treated with highly specific inhibitors of ADAM10 and ADAM17 in the presence and absence of PMA (30 ng/ml) for 4 h as described for panel A. Medium supernatants were analyzed by Western blot for the presence of meprin β (left panel). The individual lanes as shown were obtained from the same Western blot. Western band intensities were evaluated by densitometry normalized to those observed in medium supernatant of the corresponding untreated clones (right panel). Error bars represent S.E., n = 3, *, p < 0.05, ****, p < 0.0001. C, untransfected HEK293 cells were tested for the presence of ADAM9, ADAM10, and ADAM17 mRNA by RT-PCR using human ADAM9-, ADAM10-, and ADAM17-specific primers as described under “Experimental Procedures.” neg. control, negative control.