Analysis of STAT1 Activation by Six FGFR3 Mutants Associated with Skeletal Dysplasia Undermines Dominant Role of STAT1 in FGFR3 Signaling in Cartilage (original) (raw)
Figure 2
STAT1 and ERK1/2 activation by FGFR3 mutants in HeLa, 293T and RCS cells.
HeLa, 293T and RCS cells, transfected with vectors expressing wild-type FGFR, activating FGFR3 mutants (N540K, G380R, R248C, Y373C, K650M and K650E), and kinase-inactive mutant K508M were grown for 48 hours, treated with FGF2 for 15 minutes and analyzed for given molecules by western blotting. The left and right panels represent independent experiments. Note that only K650M and K650E-FGFR3 cause strong activatory STAT1(Y701) phosphorylation whereas the other mutants cause weak (HeLa) or no activation (293T and RCS). This contrasts with ERK1/2 activation, which is induced by all six mutants in 293T and RCS cells (samples without FGF2 treatment). The P-Y701-STAT1 signal in HeLa cells was quantified by densitometry and graphed. Also note the differences in FGFR3 maturation, where HeLa and RCS cells but not 293T cells express mostly immature forms (lower arrow) of K650M and K650E-FGFR3. STAT1, ERK1/2 and ACTIN western blottings serve as loading controls. Cells transfected by GFP vector serve as negative control for transfection.