X complete protease inhibitor cocktail

The dot-blot assay was performed according to Munnik and Wierzchowiecka [97 ]. A nitrocellulose membrane with immobilized phospholipids (0.25, 0.5, 1.0 μg) was incubated in Tris-Buffered Saline with Tween® 20 Detergent (TBST) blocking solution (20 mM Tris-HCl, pH 7.5; 100 mM NaCl; 0.05% Tween-20; additionally containing 1× x cOmplete™ Protease Inhibitor Cocktail (Roche) and 3% lipid-free BSA) for 5 h at 4 °C and then incubated with respective purified GST-fusion protein (10 μg/mL) in TBST at 4 °C overnight. After extensive washing with TBST (three times for 5 min), the membrane was incubated for 2 h at room temperature with primary monoclonal mouse anti-GST antibodies (Sigma-Aldrich) diluted 1:6000 in TBST supplemented with 1% lipid-free BSA (Sigma-Aldrich). Excess of antibodies was washed out with TBST as described above, and the membrane was incubated with anti-mouse alkaline phosphatase-conjugated secondary antibodies diluted 1:30,000 in TBST buffer for 1 h at room temperature, then washed extensively in TBST (5× 5 min). The secondary antibodies bound to the blots were visualized using substrates for alkaline-phosphatase (BCIP/NBT Color Development Substrate) dissolved in reaction buffer (100 mM Tris-HCl, pH 9.5; 100 mM NaCl; 5 mM MgCl₂).

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).