The N-terminal conserved region of AF6 is sufficient for myeloid immortalization by MLL-AF6. (A) Schematic diagram showing the positions of highly conserved domains within the AF6 protein family. The degree of identity between AF6 and orthologs from Drosophila and C. elegans is indicated. RA1 and RA2 indicates RAS association domains 1 and 2, respectively; NCR, N-terminal conserved region; PDZ, PSD-95/Dlg/ZO-1 domain. (B) Schematic diagram depicting MLL, AF6, and MLL-AF6 fusion protein. ATH indicates AT hook motifs; PHD, plant homeo-domain related; TAD, transcriptional activation domain; SET, Suvar3-9/enhancer-of-zeste/trithorax motif. Total number of amino acids comprising each protein is indicated on the right. Black arrows above each protein indicate the typical position of protein fusion after chromosomal translocations. Specific AF6 protein segments fused with MLL are indicated by brackets below the schematic. (C) Myeloid immortalization assay with various retroviral constructs encoding proteins indicated on the left. Each bar represents the mean ± SD of the total number of myeloid colonies per 10⁴ plated cells derived from at least 4 replicates. (D) Western blot analysis of MLL-AF6 and AF6 proteins expressed in transiently transfected virus-producing Phoenix cells. (E) Typical morphology of cells (top; May-Grünwald-Giemsa stain) and blast colony (bottom) in the third round of the myeloid progenitor immortalization assay after transduction with MLL-AF6^NCR.