Western Blot Analysis of H9c2 Cells

To perform western blot analysis, H9c2 cells were harvested with cell lysis buffer (Mammalian Protein Extraction Reagent, 78501; Pierce Thermo Scientific, Tokyo, Japan) containing protease inhibitors (#04080-11; Nacalai Tesque Inc.) and phosphatase inhibitors (#07575-51; Nacalai Tesque Inc.) on ice for 15 min. The supernatants of protein lysates were collected after 10 min of centrifugation at 10,000 × g. The protein concentrations of cell lysates were determined using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific). The samples (5 μg) were separated on 5–20% sodium dodecyl sulfate-polyacrylamide gels (#2331830; Atto, Tokyo, Japan) and transferred onto polyvinylidene difluoride membranes (BioRad, Hercules, CA) using Trans-Blot Turbo (BioRad). After being blocked with Blocking One (#03953-95; Nacalai Tesque Inc.) for 30 min at room temperature, the membranes were washed in Tris-buffered saline containing 0.1% Tween 20 (polyoxyethylene sorbitan monolaurate, 35624-15; Nacalai Tesque Inc.) three times for 10 min and incubated with primary antibodies at 4°C overnight. The following antibodies were used as primary antibodies: monoclonal anti-rat FGF23 antibody (1:500, MAB2629; R&D Systems); polyclonal anti-goat FGF23 antibody (1:1000, ab123502; Abcam, Cambridge, UK); polyclonal anti-rabbit FGFR4 antibody (1:1000, ab119378; Abcam); polyclonal anti-rabbit FGFR4 (phospho Y642; pFGFR4) antibody (1:1000, ab192589; Abcam); polyclonal anti-rabbit furin antibody (1:1000, PA1-062; Thermo Fisher Scientific); polyclonal anti-rabbit hypoxia-inducible factor 1 alpha (HIF1α) antibody (1:1000, NB100-134; Novus Biologicals, Centennial, CO); polyclonal anti-rabbit polypeptide GALNT3 antibody (1:1000, SAB2106736; Sigma-Aldrich); and anti-rabbit α/β tubulin (1:1000, CST#2148; Cell Signaling Technology, Danvers, MA). After being washed in Tris-buffered saline containing 0.1% Tween 20 three times, the membranes were incubated with the following horseradish peroxidase-conjugated secondary antibodies: donkey anti-rabbit IgG antibody (1:5000, NA934; GE Healthcare, Bucks, UK) and goat anti-rat IgG antibody (1:10,000, NA935; GE Healthcare) for 1 h. The bands were detected by the enhanced chemiluminescent method (ECL prime; GE Healthcare or Chemi-Lumi One Ultra; Nacalai Tesque Inc.), captured using a chemiluminescence imaging system (AE-9300 Ez-capture MG; Atto), and analyzed with ImageJ Software (National Institutes of Health, Bethesda, MD).