Dup00

Serum suPAR levels were measured using quantitative enzyme-linked immunosorbent assay (ELISA) kits (DUP00; R&D Systems, Minneapolis, MN, USA) in duplicate as instructed by the manufacturer. Briefly, test wells containing serum samples, and standard wells containing a gradient concentration of a standard protein, were analysed using a microplate assay. Absorbance at 450 nm was measured using the Multiskan FC (Thermo, Waltham, MA, USA). The detection sensitivity was 33 pg/mL and the intra-assay and inter-assay coefficients of variation were < 8%. Serum syndecan-4 levels were determined using ELISA (JP27188; Immuno-Biological Laboratories, Fujioka, Japan) in duplicate as instructed by the manufacturer. Absorbance at 570 nm was measured using the Multiskan FC. The detection sensitivity was 3.94 pg/mL and the intra-assay and inter-assay coefficients of variation were < 5%. Using the standard curve, the quantities of syndecan-4 and suPAR were calculated using CurvExpert Professional 2.6.3 (Hyams Development, Madison, WI, USA).

Cells at 60–70% confluency were treated as indicated in the results section. Cells were washed in PBS and scraped in a 100 µL RIPA lysis buffer containing protease inhibitor cOmplete (Roche, Mannheim, Germany), PMSF (1 mM), and orthovanadate (1 mM). Total protein was quantified using a DC™ protein assay (Bio-Rad, Hercules, CA, USA). A total of 15 µg of proteins was separated using gradient SDS gels (4–20%, Bio-Rad) and blotted on nitrocellulose membranes by a Turbo Blot (Bio-Rad). Gene signals were detected as described before [23 (link)].
uPAR protein levels were determined by ELISA (DUP00, R&D Systems, Minneapolis, USA) according to the manufacturer’s protocol. In brief, cell lysates from 10⁵ to 10⁶ cells were 10-fold diluted in a RIPA lysis buffer, and 50 μL of cell lysates or standard was added to 100 μL of assay diluent RD1W solution. The samples were incubated for two hours at RT and washed four times with a 400 μL wash buffer. A total of 200 μL of human uPAR conjugate was added and incubated for 2 h at RT. After four washing steps, 200 μL of substrate solution was added and incubated for 30 min at RT protected from light before adding 50 μL of stop solution. The optical density was measured at 450 nm with a reference of 540 nm on a Tecan reader Infinite 200 Pro. uPAR concentrations were calculated for 10⁶ cells.