CM was treated with non-reducing Western blot sample buffer and separated by SDS-PAGE (10% polyacrylamide gel containing 0.1% gelatin). The gels were washed twice with 2.5% Triton-X-100 for 15 min followed by an overnight incubation at 37°C in substrate buffer (10 mM Tris, pH 8.0, 10 mM CaCl2, 1 μM ZnCl, 150 mM NaCl). Subsequently the gel was stained with 0.5% Coomassie blue in acetic acid:isopropanol:dH2O (10:30:60) and pictures were taken after destaining with dH2O. In order to determine total protease activity in CM a sterile 6 mm filter disk was added on skim milk agar (1.5%) and 10 μl of CM was added. Following incubation at 37°C the diameter of clearance (as an indication of protease activity) was measured.