Mab143

Conditioned media were concentrated by TCA precipitation and solubilized in Laemmli sample buffer. An equal amount of proteins were separated under reducing conditions on a 10% or 12% polyacrylamide gel. Resolved proteins were transferred onto a Hybond-C nitrocellulose membrane (GE Healthcare, München, Germany) in a semidry transfer system. The transfer efficiency was assessed by staining the membrane with Ponceau Red (Sigma-Aldrich, Diesenhofen, Germany). Membranes were blocked for 1 h with 5% milk powder and incubated overnight at 4 °C with the primary antibody. Primary antibodies used were anti-ADAM9, anti-decorin, (AF949 and MAB143, respectively, all from R&D Systems, Wiesbaden, Germany), anti-collagen type I (1:500; 20315035505 from Quartett, Berlin, Germany), anti-fibronectin and anti-actin (F3648 and A2668, both 1:1000; from Sigma Aldrich, Diesenhofen, Germany), and anti-MMP-13 (sc-30073, from Santa Cruz, Heidelberg, Germany; 1:1000), anti-MMP-14 (1:500) [18 (link)]. After washing, membranes were incubated with HRP-labeled secondary antibody (from Dako, Hamburg, Germany) for 1 h at room temperature. Bound secondary antibodies were detected by ECL^® system (Thermo Scientific, Bonn, Germany) and the membranes exposed to X-ray films (Thermo Fisher, Karlsruhe, Germany).

Recombinant DCNs were analyzed on SDS/PAGE on 4–20% acrylamide gradient gels. The gels were either stained with Coomassie Blue or used to transfer the proteins to a PVDF membrane and immunoblots were performed either with mouse monoclonal anti–6-histidine tag antibody (clone 18,184; Novus Biologicals) or mouse monoclonal anti-human DCN (MAB143, R&D Systems) antibodies. The primary antibodies were detected by goat anti-mouse IgG–HRP (Bio-Rad) and then developed using ECL plus a chemiluminescence reagent (Amersham Biosciences).