Bms249 4

Venous blood samples were collected from 24 healthy volunteers (control) and 120 CRF patients who represented 5 different stages (n=24 in each stage) according to their glomerular filtration rates (GFR): 1 (GFR ≥ 90 mL/min/1.73 m²); 2 (60 < GFR< 89 mL/min/1.73 m²); 3 (30 < GFR< 59 mL/min/1.73 m²); 4 (15 < GFR< 29 mL/min/1.73 m²); and 5 (GFR < 15 mL/min/1.73 m²) 7 (link). All patients were diagnosed with CRF and received therapy in the Department of Nephrology, Jiangxi Provincial People's Hospital, during 2012-2016, and their basic characteristics are summarized in Supplementary Table 1. Blood samples were kept in K2EDTA-coated tubes (BD, Franklin Lakes, NJ, USA, #367835) until use. All participants signed a consent form reviewed and approved by the ethical board of Jiangxi Provincial People's Hospital in China. The serum concentration of TGF-β was determined by an ELISA kit (ThermoFisher Scientific, #BMS249-4).

Jurkat T cell CM was produced and collected as described above. IFN-γ and TGF-β were measured in CM using ELISA (Thermofisher Scientific EHFING and BMS249-4 respectively) according to manufacturer’s instructions.

ELISA kits were used to determine the levels of human RBP4 (KE00056, Proteintech, China), human LBP (KE00134, Proteintech, China), human TGF-β1 (BMS249-4, Thermo Fisher Scientific, USA) in serum samples or cell culture supernatants, following the manufacturer’s instruction. Signal detection was performed using spectrophotometers (Thermo Fisher Scientific, USA) at 450 nm.

Serum levels of IL-6, TGF-α, and TNF-α were determined using premixed multiplex human magnetic Luminex assays (R&D, #LXSAHM-11) according to the manufacturer’s instructions. Data acquisition was performed using the Bio-Plex MAGPIX Multiplex reader (BioRad Laboratories, Hercules, CA, USA), which uses Luminex fluorescent-bead-based technology (Luminex, Austin, TX, USA). Serum levels of TGFβ1 (EBioscience, San Diego, CA, USA, #BMS249/4) and IL-15 (Thermo Scientific, Waltham, MA, USA, #BMS2106) were evaluated with enzyme-linked immunosorbent assay (ELISA, Ratavartijankatu, Finland). The optical density was measured spectrophotometrically at a wavelength of 450 nm according to the manufacturer’s instructions. All analyses were performed in duplicate. A 4-PL standard curve was created using MyCurveFit Beta Software (https://mycurvefit.com/, accessed on 11 April 2023).

Samples were centrifuged at 15,000 g for 5 min at 4°C prior to analysis. NRP1 ligands were quantified in supernatants using ELISAs following manufacturer’s instructions; VEGF‐A (DVE00,. R&D Systems), SEMA3A (LS‐F29822, LSBio), TGF‐β (BMS‐249‐4, Thermo Fisher Scientific Inc.), PGF (EHPGF, Thermo Fisher Scientific Inc.), and PDGF‐BB (BMS2071, Thermo Fisher Scientific Inc.)