Figure 4.

Anatomic expression profile of Klotho family proteins and FGF binding protein (FGFBP1), and Lctl activity analysis. A, qPCR data are presented as the mean of triplicate measurements ± sd for tissues from C57/Bl6 mice. B, Lysates of 293 cells transfected with expression vectors for different FGFR isoforms and Flag-tagged Lctl were immunoprecipitated with Lctl using anti-Flag antibody and probed for either FGFRs using anti-V5 antibody or for Flag. Antibodies used for immunoprecipitation (ip) and immunoblotting (i.b.) are indicated. C, HEK293 cells were transfected with an expression plasmid for Flag-tagged Lctl and treated with FGF19 (1000 ng/ml), FGF21 (1000 ng/ml), FGF23 (500 ng/ml), or vehicle alone. Phosphorylated ERK (pERK), total ERK (tERK), and Flag-tagged Lctl were measured by immunoblotting. D, HEK293 cells were transfected with expression plasmids for Flag-tagged Lctl or β-Klotho and treated with FGF19 or FGF21 at the indicated concentrations. Phosphorylated ERK (pERK), total ERK (tERK), β-Klotho, and Flag-tagged Lctl were measured by immunoblotting.