Gtx 101250

Human recombinant DCN (0.5 µg) and human TGF-β1 from platelets (0.225 µg; T1654-1UG, Sigma Aldrich) were diluted in 500 µL washing buffer (0.1 M NaCI, 0.05 M Tris/HCl pH 7.4 containing 0.04% Tween-20 and 1% BSA). The following agitation was performed on a rotisserie mixer overnight. For Co-IP, 10 µg of the DCN antibody (GTX 101250, Genetex, USA) was incubated with the protein mix for 24 hours. No primary antibody was added to the negative control. The protein complexes with and without the DCN antibody were mixed with protein A magnetic beads (LSKMAGA02, Millipore) on the rotisserie mixer for 2.5 hours at 4 °C, and another 30 min at RT. Using a magnetic stand (LSKMAGS08, Millipore), the magnetic beads were removed and the protein complexes were eluted from the beads. In a next step, the protein complexes were denatured in 30 µL of 1x Roti-Load (K929.2, Carl Roth, Germany) at 90 °C for 10 min. Specific protein bands were detected after SDS-PAGE and Western blot as described before.

DCN samples were mixed with 4x Roti-Load (Carl Roth, Germany) and denatured at 90 °C for 5 min for SDS-PAGE. The samples were run on a NuPAGE®Novex 4–12% Bis-Tris 1.0 mm 12 well gel (NP0322BOX, Life Technologies, USA). For band identification, SeeBlue® Plus2 Pre-Stained Standard (LC5925, Life Technologies) was used. Using an electrical field (30 V, 60 min) in the XCell II™ Blot Module (Life Technologies), the proteins were blotted to a nitrocellulose membrane (Whatman, UK). Proteins were visualized by incubating the membrane for 5 min in a Ponceau-Red solution (Sigma Aldrich, USA). After imaging, the membrane was discolored in a 0.1 M NaOH solution for further specific protein detection. The membrane was blocked with 5% skim milk powder (Sigma-Aldrich) in TBS-T and then incubated with a DCN antibody (1:2000; GTX 101250, Genetex, USA) overnight at 4 °C. After washing with TBS-T, the membrane was incubated with the secondary goat-anti-rabbit IgG H&L (HRP) antibody (1:4000 in TBS-T with 5% skim milk powder; ab6721, Abcam, UK). After 1 h incubation at RT, the membrane was washed and a SuperSignal West Dura Extended Duration Substrate (Thermo Scientific, USA), was applied onto the membrane and the chemiluminescence was imaged using the Luminescent Image Analyzer LAS-1000 plus (FujiFilm, Japan).