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. 2014 Mar 26;111(15):5520–5525. doi: 10.1073/pnas.1402218111

Fig. 2.

Fig. 2.

Fam20C phosphorylates FGF23 in mammalian cells (A) Protein immunoblotting of trichloroacetic acid precipitates from conditioned medium of control and Fam20C KO cells (C5, clone 5; C9, clone 9) using an affinity-purified rabbit anti-Fam20C polyclonal antibody. GAPDH from cell extracts is shown as loading control (Lower). (B) Control and Fam20C KO cells were metabolically labeled with 32PO43− and transfected with V5-tagged OPN. Protein immunoblotting of V5-immunoprecipitates from conditioned medium (Top) and autoradiography depicting 32P incorporation into OPN (Middle). Fam20C protein levels in conditioned medium are also shown (Bottom). (C) Protein immunoblotting of V5-immunoprecipitates from the conditioned medium of U2OS cells expressing V5-tagged FGF23. V5-immunoprecipitates were treated with O-glycosidase and α-(2→3,6,8,9)-neuraminidase to remove O-linked glycosylation or PNGase F to remove N-linked glycosylation. (D) Control and Fam20C KO cells were metabolically labeled with 32PO43− and transfected with V5-tagged FGF23. Protein immunoblotting of V5-immunoprecipitates from conditioned medium (Top) and autoradiography depicting 32P incorporation into FGF23 (Middle). Intact FGF23 and C-terminal fragments are shown. Fam20C protein levels in conditioned medium are also shown (Bottom). IB, immunoblotting; IP, immunoprecipitation.