Western blot analysis

RAW264.7 macrophages or mouse peritoneal macrophages from wild-type and Hyp mice were cultured in 6-cm dishes as described above. Macrophages cytoplasmic protein was isolated by using an M-PER Cytoplasmic Extraction kit (Thermo Scientific, Rockford, IL, USA). Samples were quantified and stored at −80 °C until use. For electrophoresis, samples were prepared by mixing 3× SDS loading buffer (Cell Signaling) with 1× DTT. About 50 μg of protein were loaded onto NuPAGE 4–12% Bis-Tris Gel (Invitrogen, Carlsbad, CA, USA). Proteins were separated at 150 V for 60 min and transferred to nitrocellulose membrane (Invitrogen). Membranes were blocked with Superblock blocking buffer in TBST (Thermo Scientific) for 30 min and then incubated with primary antibody (FGF-23, ERK1/2, pERK1/2, 1 : 1000; Cell Signaling Technology. Klotho antibody 1 : 1000, a gift for Gwen King at UAB) with gentle agitation overnight at 4 °C. After three washes with TBST (15 min once and 2 × 5 min), membrane was incubated with secondary antibody in Superblock blocking buffer at room temperature for 1 h. Membrane was then washed four times (15 min and 3 × 5 min) and subjected to ECL (Thermo Scientific) and analyzed with the FOTO/Analyst Luminary/FX imaging workstation (FOTODYNE INCORPORATED, Hartland, WI, USA). Western blot using GAPDH antibody (Santa Cruz Biotechnology, Dallas, TX, USA) was used as internal control of protein loadings.