Axl is constitutively active in primary human AML blasts. (A) AML blasts from six FLT3-ITD⁺ patients (P1 to P6) were subjected to immunoblot to detect phospho-Axl and Axl (top). Actin was used as a loading control. Peripheral blood mononuclear cells were obtained from a normal, healthy donor by Ficoll gradient centrifugation and were used as a negative control. Numbers under each row indicate the ratio of the intensity of each band (phospho-Axl or Axl) relative to that of the actin band. Cells from panel A were analyzed for surface expression of Axl by flow cytometry using an antibody against Axl (bottom). A nonreactive isotype antibody was used as a negative control. (B) AML blasts from 2 patients possessing FLT3-ITD mutations (P3 and P5) and from 3 patients harboring FLT3-WT (P7 to P9) were subjected to immunoblot to detect phospho-Axl and Axl. CD34⁺ cells from a normal, healthy donor were used as a negative control. (C) AML blasts from 2 patients possessing FLT3-ITD mutations (P1 and P2) and from 3 patients harboring FLT3-WT (P7 to P9) were subjected to immunoblot to detect phospho-Axl, phospho-FLT3, and FLT3. PBMC, peripheral blood mononuclear cell.