Fig 1: ALK ECD cleavage is mediated by MMP-9 whose inhibition suppresses cell migration(A) WB analysis of ALK expression in lysates and CM of NGP cells treated with the broad-spectrum protease inhibitor GM6001 and the MMP-9/13 inhibitor CAS 204140–01-2 at the indicated doses, for 12 h. Untreated (U) or DMSO-treated cells were used as controls. The 8G7 N-terminal anti-ALK antibody was used to detect the shed fragment in CM and a C-terminal anti-ALK antibody used for the cell lysates in this and subsequent panels. ß-actin is used as a loading control throughout.(B) WB analysis of ALK expression in NGP cells treated with the MMP-9 (CTK8G1150) and MMP-3/12/13 (MMP408) inhibitors (2 µM × 12 h).(C) WB analysis of ALK expression in the indicated NB cell lines treated with or without CTK8G1150 (2 µM × 12 h).(D) WB analysis of MMP-9 and ALK expression in NGP and BE (2)-C NB cells expressing either a control shRNA (shGFP) or two different shRNAs against MMP-9.(E) Crystal-violet-stained images of NGP and IMR-5 NB cells treated with CTK8G1150 (2 µM) or DMSO for 16 h and then subjected to transwell migration assays for 48 h (left). Scale bar, 100 µm. Quantification of migration (right) reported as means ± SD; **p < 0.01, n = 4.See also Figure S7.
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