Fig. 5.

TIMP-1 in MDA-MB-231 conditioned medium regulates KLK7 activation of proMMP-9. (A) MDA-MB-231 conditioned medium containing 50 mM EDTA was incubated at 37°C for 4 hours with no additions (lane 1), with 1 pmol proMMP-9 (lane 2), with 1 pmol proMMP-9 and thermolysin (lane 3), or with 1 pmol proMMP-9 and 0.2 pmol thermolysin-activated KLK7 (lane 4). As a positive control, 1 pmol proMMP-9 was incubated with 0.2 pmol thermolysin-activated KLK7 in SFM containing EDTA at 37°C for 4 hours (lane 5). At the end of the incubation period, the samples were analyzed by gelatin zymography. MDA-MB-231 conditioned medium was immunodepleted with a TIMP-1 antibody (B) or control goat IgG (C). The immunodepleted conditioned medium was incubated with the additions indicated in (A) at 37°C for 4 hours prior to analysis by gelatin zymography (upper panels) or western blot for TIMP-1 (lower panels). As a positive control, 1 pmol proMMP-9 was incubated with 0.2 pmol thermolysin-activated KLK7 in SFM containing EDTA at 37°C for 4 hours (lanes 5). The truncated MMP-9 (tMMP-9) gelatinolytic products are denoted by the arrowheads and thermolysin-directed proteolysis (therm) is indicated by the arrow. Sizes of the protein markers (kDa) are indicated on the left.