Fig 2: Loss of NHR2 disrupts ETO association and dimerization of the CBFA2T3–GLIS2 complex and abrogates leukemogenesis in vivo.A–E Co-immunoprecipitation using 293T cells transfected with empty vector (MIG), wild-type fusion, or a fusion containing one or more of the mutations outlined in Fig. 4A. All immunoprecipitations were repeated twice, representative blots are shown. Source data for all blots are provided as a Source Data file. A Immunoprecipitation of ETO followed by staining with the TY-1 tag to detect CBFA2T3-GLIS2 and the reciprocal immunoprecipitation of CBFA2T3-GLIS2 via the TY-1 tag followed by staining for ETO is shown. B TY-1-tagged wild-type CBFA2T3-GLIS2 and one of three FLAG-tagged mutant constructs were co-transfected into 293T cells to evaluate the ability of the mutant constructs to dimerize with the wild-type fusion. Immunoprecipitation by TY-1 followed by staining for FLAG and the reciprocal immunoprecipitation of FLAG followed by staining for TY-1 is shown. C 293T cells were co-transfected with two mutant constructs, one FLAG-tagged and one TY-1-tagged, to verify the results shown in (B). Immunoprecipitation of TY-1-tagged NHR2 deletion mutant followed by staining for FLAG again revealed a reduction of dimerization. D TY-1-tagged wild-type and mutant fusion constructs were transfected into 293T cells. Cells were immunoprecipitated for CtBP1 followed by staining for the fusion via TY-1 and the reciprocal immunoprecipitation of the fusion followed by staining for CtBP1. E TY-1-tagged wild-type and mutant fusion constructs were transfected into 293T cells. Cells were immunoprecipitated for p300, followed by staining for the fusion via TY-1, and the reciprocal immunoprecipitation of the fusion followed by staining for p300. F, G Transplantation of immunodeficient NSG-SGM3 mice with primary megakaryoblasts transduced with the mutant fusion constructs (N = 5 per mutant construct, N = 9 wild type in panel F and 12 wild type in panel G). p < 0.0001 for NHR2 deletion and NHR1-2 (DL380,487AS) mutant constructs compared to wild type. p-values determined by log-rank test. Source data are provided as a Source Data file.