AMN107 inhibits FIP1L1-PDGFRα in vitro. (A) Dose-dependent effects of AMN107 on growth of FIP1L1-PDGFRα–transformed Ba/F3 cells. Ba/F3 cells stably transduced with the indicated constructs were treated with AMN107 for 48 hours in the absence of IL-3, and the proliferation of treated cells relative to untreated cells was determined. MSCV-GFP–transduced control cells were incubated in the presence of IL-3. Values reflect the mean of 3 samples; error bars show standard deviation. F/P indicates FIP1L1-PDGFRα. (B) Analysis of phosphorylation of FIP1L1-PDGFRα after AMN107 treatment. Ba/F3 cells transformed with FIP1L1-PDGFRα or FIP1L1-PDGFRα T674I were incubated with increasing concentrations of AMN107 in the absence of serum and IL-3. Whole-cell lysates were prepared for Western blotting with anti–phospho-PDGFRα and anti-PDGFRα. (C) Analysis of phosphorylation of Stat5, a downstream effector of PDGFRα, after AMN107 treatment. Ba/F3 cells transformed with FIP1L1-PDGFRα or FIP1L1-PDGFRα T674I were treated with increasing concentrations of AMN107 in the absence of serum and IL-3. Whole-cell lysates were prepared and analyzed for phosphorylation of Stat5, which is induced by IL-3 or by activated kinases that confer IL-3 independence. Western blotting was performed with anti–phospho-Stat5 and anti-Stat5b.