MMP-2 gelatin zymography and Western blotting

Following incubation of the cells (1×10⁵/well of a 48-well plate) in serum-free medium (150 μl), the status of MMP-2 was analyzed by gelatin zymography of the medium aliquots (15 μl) using precast 10% acrylamide gels co-polymerized with 0.1% gelatin (Life Technologies) as described previously [53]. To stimulate the MMP-2 activation, HT1080 cells (1×10⁵) were stimulated for 24 h using phorbol 12-myristate 13-acetate (50 ng/ml) with or without the presence of the Fab antibodies (20-200 nM), TIMP-1 (1,000 nM), TIMP-2 (100 nM) or GM6001 (1,000 nM). We also used the B16F1-mMT1 cells that expressed the murine MT1-MMP and the respective control B16F1-mock cells transfected with the original plasmid alone. In the latter, cells (1×10⁵) were seeded for 24 h in DMEM-10% FBS in wells of a 48-well plate. Cells were replenished with fresh DMEM (150 μl) containing purified proMMP-2 (5-10 nM) alone or jointly with the Fab antibodies (25-200 nM) or GM6001 (1,000 nM). In 18 h, the medium aliquots (15 μl) were analyzed by gelatin zymography, while cells were washed with PBS and then lysed in TBS containing 50 mM N-octyl-β-D-glucopyranoside, 1 mM phenylmethylsulphonyl fluoride, 10 mM EDTA, and a protease inhibitor cocktail set III. Insoluble material was removed by centrifugation (14,000×g; 30 min). The supernatant aliquots (5 μg total proteins) were separated by electrophoresis in a 4–12% gradient NuPAGE-MOPS gel (Life Technologies) and analyzed by Western blotting with the MT1-MMP AB8345 antibody followed by the secondary HRP-conjugated antibody (Jackson ImmunoResearch; West Grove, PA) and a SuperSignal West Dura Extended Duration Substrate kit (Thermo Fisher Scientific). Where indicated, the images were digitized and the intensity of the bands was quantified using ImageJ software. These data were used to measure the zymogen:activation intermediate ratio of MMP-2 expressed as a percentage of the zymogen and the activation intermediate each related to their combined total amount.