Western blots, showing the expression and activation of wild-type FGFR3 and the A391E mutant, as a function of ligand concentration.

HEK 293 T cells were cultured for 24 hours following transfection, and starved in serum-free medium for 24 hours before stimulation with different concentrations of fgf1. Because of the large number of samples that had to be compared, the lysates in each independent experiment were loaded onto two gels, and the proteins in the two gels were transferred simultaneously onto a blotting membrane and incubated with antibodies as described previously [20]. FGFR3 expression was probed using anti-FGFR3 antibodies, and FGFR3 activation was probed using anti-Y653/4 antibodies. The latter antibodies are specific for two phoshorylated tyrosines in the activation loop of the FGFR3 kinase. Two bands are observed in each case, one corresponding to the 120 kDa immature FGFR3 located in the ER/Golgi, and a second one corresponding to the mature 130 kDa form located predominantly in the plasma membrane. Here we quantify and process only the 130 kDa bands.