The RA1 domain within the AF6 NCR mediates myeloid immortalization by MLL-AF6. (A) Sequence alignment of the RA1 domains of AF6 family proteins. Dark shading defines residues of identity or similarity. Amino acid numbers are indicated on the left. The positions of conserved residues targeted for mutation are highlighted above the sequence. The conserved secondary structures are depicted above the sequence, where filled arrows represent β sheet and the filled box represents α helix. Brackets below indicate the RA1 amino acids present in deletion constructs identified on the left. (B) Myeloid immortalization assay resulting from transduction of various MLL-AF6 mutant proteins shown on the left. Each bar represents the mean ± SD of the total number of myeloid colonies per 10⁴ plated cells (≥ 4 replicates). (C) Western blot analysis of MLL-AF6 fusion proteins expressed in transiently transfected virus-producing Phoenix cells. (D) Immunoprecipitation–-Western blot analysis shows that only the intact MLL-AF6^RA1 is capable of coprecipitating HA-tagged RasV12 in transiently transfected 293 cells. (E) Myeloid immortalization assay with retroviral constructs encoding MLL-AF6^RA1 point mutants indicated on the left. Each bar represents the mean ± SD of the total number of myeloid colonies per 10⁴ plated cells (≥ 4 replicates). (F) Western blot analysis of MLL-AF6 mutant proteins expressed in transiently transfected virus-producing Phoenix cells.