MLL-AF6 modifies AF6 localization from cytosol to nuclear. (A) AF6 colocalizes with RAS (merged) in cytosol of healthy bone marrow (HBM) cells: (left) immunofluorescence of HBM primary cells stained with DAPI and respective antibodies against AF6 and RAS (×20 zoom). (B) (Top) Western blot analysis (WB) of AF6 and RAS expression in cytoplasmic (Cyt) and nuclear (Nu) cell extracts. (Bottom) coimmunoprecipitation (IP) of AF6 and RAS in HBM cells. Total lysates (To) were used as positive controls; negative controls (−). (C) (Top) Nuclear localization of AF6 in ML2 and SHI-1 cell lines by immunofluorescence (AF6 red, nuclei stained with DAPI in blue, ×20 zoom). (Middle) WB of AF6 and RAS expression in total (To), cytoplasmic (Cyt) and nuclear (Nu) cell extracts; anti-HDAC1 and anti-ACTIN were used as endogenous controls for nuclear and cytoplasmic proteins, respectively. (Bottom) coimmunoprecipitations (IP) between RAS and AF6 showed no interaction between the 2 proteins in neither of t(6;11) leukemic cell lines. (D) Active RAS-GTP levels in ML2, SHI-1, and HBM cells; positive control (+). Vertical lines have been inserted to indicate repositioned gel lanes.