Fig. 1

Cleavage of N-cdh by MMP-7. In (a) recombinant N-cdh (2.5 μg) was incubated with or without MMP-7 for 2 h at 37 °C. Digestion products were resolved by polyacrylamide gel electrophoresis, transferred to nitrocellulose, and then stained with Coomassie Blue (a). MMP-7 was used at increasing concentrations of 0.5, 1, and 1.5 μg/ml. In (b and c), a similar experiment was performed with MMP-2 or ADAM-10, respectively. While we do not observe appreciable cleavage with MMP-2 (b), as indicated by the arrow (c), ADAM-10 generated cleavage fragments of approximately 90 kDa. MMP-2 and ADAM-10 digests were run on the same gel for lanes shown in each figure but separated from other lanes for clarity. In (d), we demonstrate that the hydroxamate MMP inhibitor GM-6001 (GM) prevents MMP-mediated N-cdh cleavage, as expected, and that DMSO, in which GM is solubilized, does not impair generation of cleavage fragments including that observed at approximately 90 kDa (arrow). In (e), the integrity of N-cdh was also examined by Western blot using lysates from simultaneously prepared control and MMP-7-treated B35 cell culture wells. As indicated, time points at 1 and 6 h following treatment were examined in treated or control culture lysates. A reduction in full-length protein is observed within 1 h of MMP-7 treatment