Fig. 6. MEKK1-deficient fibroblasts show reduced calpain activity, and calpain inhibition mimics MEKK1 deficiency. In vivo calpain activity was assessed in fibroblasts using the cell-permeable, fluorescent calpain substrate SLLVY-AMC (A and B) and by anti-spectrin or anti-talin immunoblotting (C). (A) MEKK1+/+ cells in the presence and absence of the calpain inhibitor PD150606 (50 µM), MEKK1–/– and MEKK1 add-back cells were used for measurement of calpain activity. (B) FAK+/+ and FAK–/– cells were used to measure calpain activity as in (A). (C) The anti-spectrin and anti-talin immunoblots were stripped and reprobed with anti-m-calpain antibodies to verify protein levels. The immunoblots are representative of at least three independent experiments. (D) Wild-type fibroblasts grown to confluency on coverslips were pre-treated for 1 h with 50 µM PD150606 (left panel) or 2 µM GM6001, a matrix metalloproteinase inhibitor (right panel), and then analyzed for migration using the in vitro wound healing assay following a razor swipe in the continuous presence of inhibitor. Results are representative of at least three independent experiments for each set of experiments in (A–D).