Gelatin zymography.

MMP2 and MMP9 activities were assayed by gelatin zymography. Samples were electrophoresed on an 8% or 10% SDS-polyacrylamide gel copolymerized with 1 mg/ml gelatin (0.1%). The gels were then rinsed briefly with water and then twice for 20 min in 2.5% Triton X-100, interspersed with 1- to 5-min washes in water. The gels were incubated for 4 to 5 days in developing buffer (50 mM Tris-HCl, pH 7.6, 200 mM NaCl, 10 mM CaCl₂, 0.02% Brij 35, 0.02% NaN₃) to enable gelatin digestion by the gelatinases. The gels were then stained in 0.125% Coomassie blue for 1 h and destained in 10% acetic acid overnight. Gels processed in parallel in developing buffer containing 5 mM EDTA had no MMP activity, as expected (MMP activity is calcium and zinc dependent [12]) (data not shown). Images were acquired using an AlphaImagerHP system (Alpha Innotech) and inverted (black/white) in Adobe Photoshop or acquired from a ChemiDoc XRS (Bio-Rad). Quantification was performed with spot density analysis using AlphaEaseFC software (Alpha Innotech) or ImageJ (42), respectively, for the two image acquisition methods. The gelatin zymography standards were MMP9 (Chemicon and R&D Systems) and supernatant from human HT1080 cells (ATCC). There are multiple isoforms of MMP9 detectable by zymography (43), in which the development step activates both prepro- and pro-MMP9 forms (44). We saw heterogeneous high-molecular-mass bands (e.g., ∼120 to 130 kDa, higher than the 105-kDa murine proMMP) when large amounts of commercial recombinant murine MMP9 (R&D Systems; 909-MM) were loaded and/or when gel development times were long (data not shown). These bands likely represent a multiply glycosylated form of murine MMP9 (43; E. Candelario, personal communication). Similar bands can be seen in the human HT1080 marker lanes (Fig. 1, ​,2,2, ​,7,7, and ​and9,9, M3) when large amounts or long developing times were used.