(A) Immunoprecipitation analyses in 293T cells transfected with OTT-MAL and OTT-MALΔRRM mutant show interaction between OTT-MAL and RBPJ through the N-terminal region of OTT (see B). STAT5 antibody was used as a nonspecific control. (B) Schematic representation of OTT, MAL, OTT-MAL (OM), and OM mutants. (C) 293T cells were transfected with the indicated constructs. Luciferase assays were performed 48 hours after transfection. Mean ± SD light intensity of 3 independent experiments is shown. Inset, expression analysis of the different constructs in 293T cells by Western blot (anti-HA antibody). (D) TNR mice (GFP under the control of RBPJ-responsive elements) were crossed to OM animals, and GFP fluorescence intensity was analyzed in lineage-negative hematopoietic progenitors from WT, TNR, and OM+TNR mice. (E) RNA was extracted from lineage-negative cells of OM and WT littermate control animals and analyzed by quantitative RT-PCR for Notch target genes compared with Gapdh. Mean ± SD of 3 independent experiments is shown. (F) Immunoprecipitation analyses of 6133 cell lysate indicate interaction between RBPJ and OTT-MAL in AMKL cells. (G) Chromatin immunoprecipitation assays were performed with 6133 cells and Ba/F3 cells and analyzed by quantitative PCR using primers surrounding a RBPJ binding site in the endogenous promoter of the Hes1 gene. Mean ± SD of triplicate quantitative PCR from a representative experiment are shown. (H) 6133 cells were transduced with a retrovirus expressing a dnRBPJ mutant. GFP-positive cells were flow sorted, and proliferation was monitored using trypan blue exclusion assay. Mean ± SD relative to noninfected cells is represented (n = 3).