FGF21 Activates Preferentially Adipose FGFR1-KLB were stripped by using a solution of 40% methanol and 2% acetic acid for 30 min. [55,73]. Experiments were repeated at least three times with samples from at least three independent preparations. Cell surface receptor binding, cross-linking and binding constant determination 125Iodine labeling of FGF1 and subsequent heparin-sepharose purification were as described [73]. Active recombinant His- tagged FGF19 after iodination was purified by Ni-Chelating sepharose chromatography following the established procedure [4]. Binding was done under specific conditions designed to support specific binding [73] on the surface of T-Rex 293 cells expressing different combinations of FGFR1, FGFR4 and KLB. Specific binding of 125I-FGF to the FGFR ectodomain was distinguished and extracted from non-specific binding, and confirmed by covalent affinity crosslinking as described [4,74]. 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J Cell Physiol 210: 1–6. PLoS ONE | www.plosone.org 11 March 2012 | Volume 7 | Issue 3 | e33870