Fig 1: D11 inhibits proliferation of FGFR3-expressing cells. (A-D) D11 blocks ligand-dependent viability of FGFR3b- or FGFR3c-expressing BaF3 cells. Cells were cultured in growth medium without FGFs (no ligand), with 3.7 nM FGF1 or FGF9 and 15 µg/ml heparin, alone, or in combination with control antibody (hIgG) or D11. Cell viability was measured with CellTiter-Glo after 72 h incubation with antibodies. (E) D11 inhibits proliferation of bladder cancer cell line UMUC-14 harboring the FGFR3 S249C mutation. UMUC-14 cells were incubated with increasing amount of control antibody (hIgG) or D11 for 72 h. Cell viability was measured by CellTiter-Glo. (F,G) Western blot showing incubation with D11 causes FGFR3 protein loss. UMUC-14 cell lysates after 72 h incubation with control antibody (hIgG) or increasing amounts of D11 were analyzed by SDS-PAGE and probed with anti-FGFR3 and anti-tubulin antibodies (F). Bands were detected and quantified using a Fujifilm LAS-4000 Luminescence Image Analyzer (G). A-D, n=3; E-G, n=2. Error bars represent s.d.
Fig 2: D11 binds to FGFR3 receptor and inhibits ligand binding in vitro. (A) Selective binding of D11 to FGFR3. Human FGFR1-4 Fc fusion proteins were immobilized and incubated with an increasing amount of D11 antibody. (B,C) Similar binding of D11 to human and mouse FGFR3b-Fc (B) or FGFR3c-Fc (C) fusion proteins. (D) Flow cytometry analysis of D11 binding to FGFR3b- or FGFR3c-expressing BaF3 cells. (E-H) Inhibition of FGF1 (E,F) and FGF9 (G,H) ligand binding by D11 to immobilized FGFR3b-Fc (E,G) or FGFR3c-Fc (F,H) fusion proteins. Specific binding was measured using SULFO-TAG-labeled FGF ligands in the presence of increasing amount of D11. A-D, n=2; E-H, n=3. Error bars represent s.d.
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