TEL-FGFR3 promotes IL-3–independent cell growth of Ba/F3. Inhibitory effect of SU5402 on TEL-FGFR3 transformants. (A) Ba/F3 cells were transfected with TF-V5 (□), ΔHLH-TF-V5 (○), or empty vector (mock; ⋄). Then, 2 × 10³ cells were plated (i) in the presence of IL-3 or (ii) in the absence of IL-3. The effects of SU5402 (iii) and AG1296 (iv) on the growth of polyclonal transformants are shown. At daily intervals, 20 μL MTS was added to the cultures and incubated for 1 hour. In (iii), □ indicates 0 μM SU5402; ○,5 μM; ▵,10 μM; , 25 μM; ⋄, DMSO; and , mock 25 μM with IL-3. In (iv), □ indicates 0 μM AG1296; ○, 1 μM; ▵, 5 μM; , 10 μM; 25 μM; ⋄, DMSO; and ⊕, mock 25 μM with IL-3. Each value is the mean of 3 experiments done in triplicate plus the SD. (B) Protein expression and autotyrosine phosphorylation of TEL-FGFR3 in polyclonal transfectants. Ba/F3 cells (2 × 10⁶) were transfected with empty vector (lane 1), ΔHLH-TF-V5 clone (lane 2), TF-V5 clone (lane 3), or TF-V5 clone and incubated in the presence of 0.1% DMSO (lane 4), 5 μM SU5402 (lane 5), 10 μM SU5402 (lane 6), or 25 μM SU5402 (lane 7) for 1 hour or with 5 μM AG1296 (lane 8), 10 μM AG1296 (lane 9), or 25 μM AG1296 (lane 10) for 1 hour. Cells were lysed, immunoprecipitated with anti-V5 antibody, and analyzed by Western blotting using antiphosphotyrosine antibody (4G10) as indicated. The same membrane was reprobed with anti-V5 antibody and showed that nearly equivalent amounts of fusion protein were loaded in each lane.