Roti load

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.

For mitochondrial OXPHOS protein expression, proteins were extracted in RIPA buffer, and protein concentration was determined with the BCA assay (Pierce). Cell lysates were then diluted in SDS-PAGE loading buffer (Roti-Load, K929.1, Carl Roth), and SDS-PAGE was performed on 20 μg of cell lysate per sample on Tris-glycine-SDS polyacrylamide gels (4561095, Biorad), then transferred to PVDF (1620177, Biorad) using standard techniques. Primary antibodies used were against the mitochondrial respiratory chain subunits (mitochondrial OXPHOS antibody cocktail containing cytochrome c oxidase subunit 2 [COX2], cytochrome b-c1 complex subunit 2 [UQCRC2], succinate dehydrogenase [ubiquinone] flavoprotein subunit B [SDHB], NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 8 [NDUFB8] and ATP synthase subunit alpha [ATP5A]; Cat. #ab110411, Abcam). Total protein detection (REVERT Total Protein Stain Kit, P/N 926-11010; Li-Cor) was used to normalize total cellular protein. Primary antibodies were detected with appropriate anti-mouse or anti-rabbit Infared IRDye 680 RD or 800 CW antibodies (Abcam). Protein band intensities were detected with the Odyssey 3.0 (Li-Cor Biosciences) and quantified using Image Lab 6.0 software (Biorad), and band intensity determined in the linear range was normalized to the band intensity of total protein.

Denaturing SDS-PAGE was performed using 10% (w/v) separating polyacrylamide/urea gels with 4% (w/v) stacking gels, according to Schägger and Von Jagow (1987) (link). The samples were denatured with Rotiload (Roth) at room temperature before loading and, after the electrophoretic separation, the gels were stained with Coomassie Brillant Blue G250 or silvered according to Switzer et al. (1979) (link). For the glycoprotein staining assay, the Pro-Q Emerald 300 kit was used according to the manufacturer’s instructions (Molecular Probes). The molecular weight of the resolved RhVI1 was estimated by plotting the retardation factor values (R_f, length of the band migration/length of the dye front) versus the logarithm of the molecular marker weights (Prestained Standard high range, Bio-Rad) using a polynomial regression curve fit.