Figure 2.

Analysis of FGFR signaling in MC3T3 cells. (A and B) Comparison of the osteocalcin FGF response element (OCFRE-luc) activity of cells transiently transfected with FGFR2c6Myc (wild type, open bars) or FGFR2c6Myc (S252W) (solid bars). (A) MC3T3 cells treated with indicated concentrations of FGF2; (B) MC3T3 cells treated with indicated concentrations of FGF7. OCFRE-luc, and pSVβ-gal were cotransfected as described in Materials and Methods. Both luciferase and β-galactosidase activities were quantified. All data were normalized to β-galactosidase activity and then plotted as fold induction over the wild type without FGF treatment. Fold induction was calculated by dividing the mean (±standard deviation) derived for each construct by the mean of the FGFR2c6Myc (wild type) without added FGF. (C and D) Phosphotyrosine analysis of MC3T3 cells stably expressing wild-type FGFR2c6myc or FGFR2c6Myc (S252W). (C) Cells treated with FGF2. (D) Cells treated with FGF7. Receptor proteins were immunoprecipitated with the anti-myc antibody and detected with antiphosphotyrosine antibody (C Upper; D Left). The expression level of receptor proteins was determined by reprobing the same blots with anti-myc antibody 9E10 (C Lower; D Right).