Anti fgf23

Osteoblasts were homogenized in 0.2 mL of lysis buffer (Beyotime, China) and kept on ice during the trial procedure. The homogenate was centrifuged at 12,000 g for 5 min at 4°C, and the supernatant was collected. Protein concentration was assayed using a BCA assay kit (Beyotime, China) according to the manufacturer's protocol. Aliquots of 18 μg of protein were separated with 7.5 to 10% SDS polyacrylamide gels (Bio-Rad, Richmond, 246 CA), and the proteins were then transferred onto a PVDF membrane (Millipore, Darmstadt, Germany) at 200 mA for 2 h in a Tris-glycine buffer with 20% anhydrous ethanol at 4°C. Membranes were blocked with the western blocking buffer (Beyotime, China) for 1 h at room temperature. The membranes were then probed with primary antibodies at 4°C with gentle shaking overnight. The primary antibodies used were anti-FGF23 (ABClonal, China), anti-ERK (CST), anti-phospho-ERK (CST, MA, USA), and anti-α-Tubulin (Beyotime, China). After being washed, the membranes were incubated with horseradish peroxidase-linked anti-rabbit secondary antibodies for 4 h at 4°C. Membranes were then visualized by exposure to Hyperfilm ECL (Beyotime, China). Films were scanned, and specific bands were quantified using ImageJ 1.43 software (National Institutes of Health, Bethesda, MD, USA). The band intensity was normalized to the α-Tubulin band in the same sample.