Figure 4.

TIMP-2 binding with MT1-MMP activates MEK1/2 upstream of ERK1/2. (a) MT1-MMP-siRNA and siRNAscr cells were incubated for 24 h in DMEM supplemented with TIMP-2 (100 ng/ml), GM6001 and U0126 (10 µM each) alone and in combination. Cell lysates were analyzed by immunoblotting for pERK1/2, total ERK1/2, pMEK1/2 and total MEK1/2. (b) Cells were incubated for 24 h in DMEM supplemented with TIMP-2 (50 ng/ml or 100 ng/ml) and GM6001 and U0126 (10 µM each) in combination or alone. Cell lysates were analyzed by immunoblotting for pERK1/2 and total ERK1/2. (c) Migration efficiency of MT1-MMP-siRNA and siRNAscr cells. Cells (1 × 10⁴) were allowed to migrate for 12 h. Where indicated, TIMP-2 (50 ng/ml) and U0126 (20 µM) alone or in combination were added. (d) Cells where incubated with TIMP-2 (50 ng/ml) and GM6001 (10 µM) alone or in combination. A 100% number denotes the migration efficiency of the untreated cells. The data are from three independent experiments performed in triplicate. *p < 0.05. These experiments were repeated three times with comparable results.