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

Fig. 1. CSF2RB interacts with FLT3.

Fig. 1

A, B Ba/F3 cells expressing CSF2RB-Flag and either FLT3 or FLT3-ITD were serum-deprived for 5 h with addition of 2 ng/ml IL-3 (5 min), 50 ng/ml FLT-ligand (5 min), 100 nM midostaurin (1.5 h), 300 nM sorafenib (1,5 h) or 100 nM gilteritinib (1,5 h) as indicated. Co-immunoprecipitations were performed using FLT3-antibody (A) or anti-flag beads capturing CSF2RB (B). Immunoprecipitates and whole-cell lysates were subjected to SDS–PAGE and western blot analysis using indicated antibodies. C, D FLT3-ITD-positive AML cell lines MV4–11 and MOLM-13 were serum-deprived for 4 h and additionally treated with 100 nM midostaurin for 1.5 h as indicated. Co-immunoprecipitations were performed using FLT3-antibody (C) or CSF2RB-antibody (D). Immunoprecipitates and whole-cell lysates were subjected to SDS–PAGE and western blot analysis using indicated antibodies. E Proximity ligation assay (1-PLA) was performed using oligo-coupled primary antibodies against FLT3 and CSF2RB. FLT3 expressing OCI-AML3 cells and FLT3-ITD expressing MV4–11 and MOLM-13 cells were fixed (surface) or fixed and permeabilized with 0.5% saponine (cellular) prior to PLA reaction. Red dots indicate the occurrence of a close FLT3: CSF2RB proximity. Nuclei were counterstained with DAPI. Representative images are shown (Zeiss 780 Meta confocal microscope; Objective NA 1.4), scale bar = 10 µm. Quantification of the PLA signals is shown as signals per cell. F Blast cells from three FLT3-ITD positive AML patients and one FLT3-ITD negative AML patient (patient 4) were isolated from peripheral blood using Ficoll density gradient. Cells were fixed (surface) or fixed and permeabilized (cellular) and PLA was performed (as described in E), p ≤ 0.0002.