Overexpression of Flt3-ITD leads to the SMRT localization from nucleus to cytoplasm. (A) Alteration of subcellular localization of GFP-SMRT by Flt3-ITD. The 293T cells were transfected with GFP-SMRT together with an empty expression plasmid (top) or Flt3-ITD expression vector (bottom) and stained with 4′6 diamidino-2-phenylindole (DAPI). (B) Schematic presentation of SMRT deletion mutants assayed for nuclear/cytoplasmic localization in response to Flt3-ITD (EGFP, enhanced GFP). (C) Biochemical subcellular fractionation of SMRT proteins. GFP-SMRT and deletion mutants were introduced into 293T cells with either an empty vector (V) or Flt3-ITD (F), as indicated. The cells were harvested and separated into nuclear and cytoplasmic fractions. Equal proportions of the nuclear and cytoplasmic fractions were separated by electrophoresis and immunoblotted for SMRT. Lighter exposures of the same blot were analyzed using NIH image 1.6 in order to quantify the levels of expression of SMRT. Relative density of cytosolic SMRT was determined as described in Figure 2. The quality of the fractionation was determined by immunoblot for nuclear RNA polymerase II and cytoplasmic β-actin. (D) Subcellular localization of GFP fusion proteins. Cells transfected with the indicated GFP-SMRT constructs in the presence (F) or absence (V) of coexpressed Flt3-ITD were visualized by immunofluorescence microscopy and the percentage of cells exhibiting nuclear or cytoplasmic SMRT expression was scored. The data represent the average percentage of 2 independent experiments of at least 200 cells per experiment. C indicates cytoplasmic; and N, nuclear.