Complete protease and phosphatase inhibitor cocktail and edta

UMR106 cells treated with or without 100 ng/ml oncostatin M for 24 h were lysed in ice-cold RIPA buffer (Cell signaling, Frankfurt, Germany) supplemented with complete protease and phosphatase inhibitor cocktail and EDTA (Thermo Fisher Scientific). After centrifugation at 10,000 g and 4 °C for 5 min, proteins were boiled in Roti-Load 1 buffer (Carl Roth, Karlsruhe, Germany) for 10 min. Proteins (30 µg per lane) were separated on 12% SDS polyacrylamide gels, and transferred to nitrocellulose membranes. Membranes were incubated overnight at 4 °C with rabbit anti-FGF23 antibody (diluted 1:1000, #BS-5768R; Thermo Fisher Scientific) or rabbit anti-GAPDH antibody (diluted 1:2000, #5174; Cell Signaling), and then with secondary goat anti-rabbit HRP-conjugated antibody (1:5000; Cell Signaling) for 1 h at room temperature. For loading control, membranes were stripped in stripping buffer (Roti-Free Stripping buffer 2.2 plus; Carl Roth, Karlsruhe, Germany) at room temperature for 30 min. Antibody binding was detected with ECL detection reagent (Bio-Rad Laboratories) and densitometric analysis was performed by using Image Lab software 6.1 (Bio-Rad Laboratories). The results are shown as the ratio of FGF23 protein to GAPDH, normalized to the control group.