Gelatin Zymography for MMP Activity

For the analysis of MMP activity, cells were treated either with vehicle or β-NEP for 12 h. Then, protein samples were prepared [51 (link)] and loaded without heating on a 10% SDS-polyacrylamide gel including 2 mg/mL gelatin from porcine skin (Sigma-Aldrich, St. Louis, MO, USA). After electrophoresis, the gels were washed in 2.5% Triton X-100 for 30 min at room temperature to allow the proteins to renature and then incubated at 37 °C overnight in the substrate buffer (50 mmol/L Tris-HCl, 200 mmol/L NaCl, 10 mmol/L CaCl₂, 1 μmol/L ZnCl₂). Proteins were stained with Coomassie Brilliant Blue R-250 solution (Sigma-Aldrich) to reveal zones of lysis, and band intensity was calculated using ImageJ software.