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. 2021 Nov 8;36(3):701–711. doi: 10.1038/s41375-021-01462-4

Fig. 4. Interaction of CSF2RB and FLT3 is independent of physiological receptor complex formation and takes place at the cytosolic site close to the membrane.

Fig. 4

A Parental Ba/F3 cells and FLT3-ITD-expressing Ba/F3 cells were serum-deprived for 4 h and left untreated or incubated with IL-3 at 2 ng/ml. Whole-cell lysates were subjected to immunoprecipitation using antibodies against CSF2RB. Immunoprecipitates and whole-cell lysates were subjected to SDS–PAGE and western blot analysis using indicated antibodies. B Mapping of the CSF2RB and FLT3 binding sites by GST-pulldown assay. Schematic diagrams show the regions used for glutathione S-transferase tagged peptides captured by glutathione-coated agarose beads. In vitro translated intracellular domains of FLT3-ITD and CSF2RB served as binding partners (input). Incubation was performed overnight; interaction complexes were separated by SDS–PAGE and subjected to western blot analysis using the indicated antibodies. JMD juxtamembrane domain, TKD1 tyrosine kinase domain 1, TKD2 tyrosine kinase domain 2. C CSF2RB-deficient gamma-2A cells were transduced with two different variants of CSF2RB (full-length (FL) or del461-473) and human FLT3-ITD or in combination with empty vectors, as indicated. Whole-cell lysates were subjected to immunoprecipitation using antibodies against FLT3. Immunoprecipitates and whole-cell lysates were subjected to SDS–PAGE and western blot analysis using indicated antibodies.