Effects of single or multiple substitutions at tyrosine residues on FGFR3 TDII activity in Ba/F3 cells. Ba/F3 cells transduced with an empty retroviral vector were included as a negative control. (A) IL-3–independent growth of Ba/F3 cell lines stably expressing distinct FGFR3 TDII single mutants. Cells were cultured with aFGF and heparin in the absence of IL-3 and counted daily. (B) Effects of multiple mutations at tyrosine residues on FGFR3 TDII–dependent IL-3–independent growth of Ba/F3 cells. (C) Effects of diverse tyrosine mutations on FGFR3 TDII transforming activity in a cell viability assay. The relative cell viability was normalized to the viability of cells stably expressing FGFR3 TDII mutant control. Data presented are mean ± standard error (n = 3). (D) Expression of distinct FGFR3 TDII variants in stably transduced Ba/F3 cells. FGFR3 TDII proteins were detected with antibody recognizing FGFR3 C-terminal tail. Visualization of double bands in each lane is due to the 2 alternative translational start sites in FGFR3. WB indicates Western blot. (E) Mutation Y760F abolishes FGFR3 TDII–dependent phosphorylation and activation of PLCγ. PLCγ specifically phosphorylated at activating tyrosine residue (Y783) and expression of total PLCγ were detected by immunoblotting. (F) Non–activation loop tyrosine residues are dispensable for FGFR3 TDII–dependent phosphorylation and activation of MAPK. Cells were treated with 1 nM aFGF and 30μg/mL heparin for 5 minutes prior to lysis. Phospho-MAPK and total MAPK protein were examined. FGFR3 TDII stable cells without ligand treatment were included as a control.