Quantifying Kidney and Plasma Proteins

Kidney proteins were extracted as recently described (Zwischenberger et al., 2021 (link)). Plasma proteins were used directly from obtained plasma. The protein concentration of each sample was evaluated by DC Protein Assay (BioRad) according to the manufacturer’s protocol. Proteins (10 μg per lane) were resolved by SDS-PAGE gel electrophoresis using TGX stain-free gradient (4%–15%) gels, visualized using stain-free technology (ChemiDoc MP Imaging System, BioRad) and transferred to polyvinylidene difluoride (PVDF) membranes (Trans-blot Turbo Transfer System, BioRad). The membranes were blocked for 1 h in 3% nonfat dry milk at room temperature and incubated with primary antibody in 1% nonfat dry milk overnight at 4°C. HRP-linked secondary antibody incubation (anti-rabbit IgG at 1:5000, HAF008, R&D, and anti-mouse IgG at 1:5000, sc-2005, Santa Cruz Biotechnology) was performed for 1 h at room temperature followed by chemiluminescence detection (Clarity Western ECL Substrate, BioRad). Primary antibodies were as follows: Rabbit anti-mouse PAI-1 (1:1000, ab182973, Abcam), rabbit anti-mouse NGAL (1:1000, ab63929, Abcam), and mouse anti-fibrin clone 59D8 (1:1000, MABS2155, Millipore Sigma). Densitometry analysis was performed on the resulting blots using Image Lab software and bands were normalized for total protein content of each lane.