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. 2009 Jul 1;284(35):23386–23396. doi: 10.1074/jbc.M109.034090

FIGURE 2.

FIGURE 2.

Constitutive and ionomycin-stimulated collagen XVII shedding is strongly inhibited by the ADAM10-selective GI254023X. A, inhibition of constitutive collagen XVII shedding from murine primary keratinocytes by increasing concentrations of the hydroxamate inhibitors GI and GW280264X (GW). Confluent murine keratinocytes were incubated with DMSO vehicle control or with 0.5, 1, and 2 μm inhibitor for 3 h in culture medium. Western blots of the cell lysates were probed for full-length collagen XVII (CXVII) and the ectodomain (ECTO) with a mixture of MO-NC14A and Endo-2 antibodies with ERK-2 serving as loading control. The released ectodomain (ECTO) in cell supernatants was visualized using the MO-NC14A antibody. Each immunoblot is representative of three independent experiments, the results of which are summarized as mean ± S.D. in the graph in the lower panel (##, p < 0.01; ###, p < 0.001). Collagen XVII shedding is strongly inhibited by 1 μm GI, a concentration that is selective for ADAM10 over ADAM17 (34, 35). B, the constitutive and stimulated shedding of murine AP-tagged collagen XVII in transfected COS-7 cells was analyzed by AP assay. The cells were incubated for 25 min without (Co) or with 2.5 μm IM and/or 1 μm GI or the non-selective hydroxamate BB94 (BB). Data represent the mean ± S.D. (n = 3). Both constitutive and stimulated shedding (**, p < 0.01) was efficiently inhibited by 1 μm GI (##, p < 0.01; ###, p < 0.001). C, inhibition of ionomycin-stimulated shedding of collagen XVII from murine keratinocytes with GI. Western blot analysis of cell lysates probed with both MO-NC14A and the Endo-2 antibody with ERK2 used as loading control and Western blots of concentrated supernatants probed with MO-NC14A after treatment with 1 μm ionomycin for 20 min revealed strong inhibition of shedding in the presence of 1 μm GI. The graph in the panel on the right summarizes the densitometric analysis of immunoblots of the released collagen XVII ectodomain from three different experiments (mean ± S.D.; **, p < 0.01; #, p < 0.05).

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