Western Blots

Collagen hydrogels, neprilysin standards (20-50-100 ng/lane) or medium with released neprilysin (20 μl per lane) were denatured (10 min 70°C with reducing agent) and loaded onto 10% Bis-Tris polyacrylamide gel (Invitrogen) and electrophoresis was performed for 35 min at 200 V. Samples were electrotransferred onto PVDF membranes for 20 min at 25 V in a semi-dry transfer cell (Thermo Fisher Scientific). Blotting was done by using WesternBreeze Chemiluminescent immunodetection system (Invitrogen). Blots were blocked with blocking buffer for 30 min and incubated overnight on a shaker at 4°C with primary antibodies against neprilysin (abcam ab210932; 1:1000). Subsequently, blots were briefly washed and incubated with alkaline phosphatase-conjugated secondary mouse (neprilysin) for 30 min at room temperature. Afterward, the blots were briefly washed again, incubated for 15 min in CDP-Star chemiluminescent substrate solution (Roche) and visualized with a cooled CCD camera (SearchLight, Thermo Fisher Scientific).