Profiling Furin and PC Enzyme Activities on FGF23 Processing

The cDNAs coding for human furin, PC7, and PACE4, and mouse PC5A and PC5B were cloned into the pIRES2-EGFP vector (31 (link)), while the cDNA of hFGF23 was cloned in pIRES2-EGFP-V5. CHO-K1 and furin-deficient CHO-FD11 cells were cultured at 37°C and 5% CO₂ in Ham’s F-12 medium supplemented with 10% (v/v) FBS (32 (link)) and transfected with 3 µg of plasmid following the standard protocol for Lipofectamine 2000 (Thermo Fisher). For the PCs inhibition, CHO-K1 cells were transfected with cDNAs encoding for hFGF23-V5 and PCs in the ratio of 5:1 and 24 hours later pretreated for 5 hours with Dec-RVKR-CMK (50 μM; Tocris) or D6R (20 μM; Calbiochem) followed by 22 hours of treatment in serum free media. Cells supernatant was then collected in the presence of 1x complete protease inhibitor cocktail (Roche) and FGF23 processing was analyzed by SDS-PAGE (15% Tris-glycine) followed by western blot using V5 antibody (Thermo Fisher).