Ki11502 inhibits RTK and its downstream signal pathways. (A,F) Coimmunoprecipitation. EOL-1 (A) or MOLM13 (F) cells were cultured with various concentrations of Ki11502 (0.1-1 nM for EOL-1 and 0.1-1 μM for MOLM13). After 3 hours, cells were harvested and proteins were extracted. The PDGFRα or FLT3 protein was immunoprecipitated and subjected to Western blot analyses. The membrane was probed sequentially with an antiphosphotyrosine antibody (top) and an anti-PDGFRα or FLT3 (bottom). p-Ty, phosphotyrosine. (B) Analysis of ERK, Akt, and STAT5 in EOL-1 cells by FACS. EOL-1 cells were cultured with Ki11502 (0.1-1 nM) or control diluent. After 3 hours, cells were harvested, incubated with the indicated antibodies for 30 minutes at room temperature, and analyzed by flow cytometry. Results are representative of 3 experiments performed in duplicate plates. The positive population was quantified using the CellQuest software package. (C) Western blot analysis. EOL-1 cells were cultured with various concentrations of Ki11502 (0.1-1 nM). After 3 hours, cells were harvested, and proteins were extracted and subjected to Western blot analysis. The membranes were sequentially probed with the indicated antibodies. Results represent one of the 3 experiments performed independently, each giving similar results. (D,E,G) Analysis of FLT3, ERK, Akt, and STAT5 in MV4-11, MOLM13, and freshly isolated leukemia cells by FACS. MV4-11 (D), MOLM13 (E), and freshly isolated leukemia cells from patients (G, cases 1-3, Table 3) were cultured with either Ki11502 (0.5 μM) or control diluent. After 3 hours, cells were harvested, incubated with the indicated antibodies for 30 minutes at room temperature, and analyzed by flow cytometry. Results are representative of 3 experiments performed in duplicate plates.