AP Assay and FGF Binding
COS-7 cells were transfected by a standard DEAE-dextran method with 4 mg of plasmid/10 6 cells. Conditioned medium was harvested 3, 6 and 9 days after transfection and used for AP enzyme activity and to determine the relative concentration of the soluble receptors. Binding of the receptor to an immobilized heparin-FGF complex was done as previously described (Gray et al., 1996). Briefly, 10µl bed volume heparin-sepharose was mixed 1:1 with sepharose CL6B and 100 ng FGF2, FGF4 or FGF8. After adding PBS to 50 ml, the mixtures were rotated at room temperature for 1 hour, then washed twice with 100 µl PBS before the addition of receptor supernatant (800 mOD/minute). Following the addition of receptor supernatant, the mixtures were further incubated at room temperature for 4 hours, then washed three times with 200 µl 20 mM Hepes pH 7.5, 150 mM NaCl, 1% Triton X-100, 10% glycerol, and twice with 150 µl 10 mM Tris pH 7.5, 0.5 M NaCl. The receptor-FGF-heparin sepharose complex was transferred in 60 µl 10 mM Tris pH 7.5, to a well of a microtiter plate containing 60 µl of 2« AP assay buffer (2 M diethanolamine, 1 mM MgCl2, 20 mM homoarginine, 12 mM p-nitrophenyl phosphate pH 9.5). The bound receptor was quantified by measuring the initial rate of substrate hydrolysis at 405 nm and same amount of proteins was used in the binding assays.
Gray, T. E., Eisenstein, M., Yayon, A. and Givol, D. (1996). Asparagine-344 Is a Key Residue For Ligand Binding In Keratinocyte Growth Factor Receptor. Biochemistry 35, 15640-15645.
PCR Assay for FGFR3 -/- Matings
PCR for FGF9 KO mice
PCR for FGF17 KO mice
PCR assay for hGH (colII-FGFR3 mice)