IL1RAP physically interacts with cytokine receptors FLT3 and c-KIT in human AML cells. (A) Coimmunoprecipitation of HA-tagged IL1RAP in protein lysates from 293T cells transfected with IL1RAP-HA and FLT3-myc fusion vectors. Immunoblot (IB) was performed for IL1RAP and FLT3. (B) Coimmunoprecipitation of endogenous FLT3 in protein lysates from THP-1 cells. Immunoblot was performed for FLT3 and IL1RAP. (C) Fluorescence energy transfer (FRET) between IL1RAP and FLT3. THP-1 (left) or MOLM-13 (right) cells were stimulated with 200 ng/ml rhIL-1β or 100 ng/ml rhFLT3L. For C and G, cells were stained with fluorescent antibodies against the indicated proteins for flow cytometry analysis and FRET was determined for cells expressing both receptors. Data represent the mean ± SD of three independent experiments. (D) Coimmunoprecipitation of HA-tagged IL1RAP in protein lysates from 293T cells transfected with IL1RAP-HA and FLT3-ITD-myc fusion vectors. Immunoblot was performed for IL1RAP and FLT3. (E) Coimmunoprecipitation of HA-tagged IL1RAP in protein lysates from 293T cells transfected with IL1RAP-HA and c-KIT-myc fusion vectors. Immunoblot was performed for IL1RAP and c-KIT. (F) Coimmunoprecipitation of endogenous c-KIT in protein lysates from THP-1 cells. Immunoblot was performed for c-KIT and IL1RAP. (G) FRET between IL1RAP and c-KIT. THP-1 (left) or HEL (right) cells were stimulated with 200 ng/ml IL-1β or 100 ng/ml SCF. Data represent the mean ± SD of three independent experiments. *, P < 0.05; **, P < 0.01.