FGF-23 functional assays

For FGF-23-mediated activation of FGFR/α-Klotho complexes, the HEK-293 cells were transiently transfected with α-Klotho membrane or secreted form along with Elk1-Gal4 luciferase reporter system and Renilla luciferase-null as internal control plasmid by electroporation using Cell Line Nucleofector Kit R according to the manufacturer’s protocol (Amaxa, Inc., Gaithersburg, MD, USA). About 48 h after transfection, the transfected cells were treated with 1 nM FGF-23 from different macrophages for 6 h. The cells were then lyzed in 1× passive reporter lysis buffer (Promega), and luciferase activities were measured using an Autolumat Luminometer (Wallac-Berthold, Gaithersberg, MD, USA) and Promega Dual-Luciferase^® Reporter Assay System. Data represent results of at least three separate experiments.