Gelatinolytic activity of MMP-2 in the presence of PARP and MMP inhibitors. Gelatinolytic activity of 64 kDa MMP-2 by zymography was performed as previously described [5] with some modifications. Briefly, 10 ng of 64 kDa MMP-2 were electrophoresed on
PI-103 8% polyacrylamide gel containing 2 mg/ml gelatin for 60–90 min (150 V, ambient temperature). After washing with Triton X-100 (2.5% v/v, 3 × 20 min), the gels were cut and one electrophoresed protein per strip was separately incubated overnight at 37 °C in 20 ml zymography buffer in the
altitudinal gradient absence or presence of PJ-37, EB-47, 5-AIQ (10, 30, and 100 μM), and 3-AB (30, 100, and 1000 μM). For comparison, gel strips were incubated in the absence or presence of MMP inhibitors, i.e. o-phenanthroline (3, 10, and 30 μM), doxycycline (10, 30, and 100 μM) and GM 6001 (0.3, 1, and 3 μM). Gels were stained with 0.05% Coomassie blue and subsequently destained. Gelatinolytic activities were detected as transparent bands against the blue-stained background.
