The interaction between Diversin and AF9 was restricted to the nuclear compartment of the cell. (A) The interaction between Diversin and AF9 was restricted to the nuclear compartment of the cell. HEK 293T
Bleomycin Sulfate expressing V5.AF9 and FLAG9.Diversin were lysed and separated into nuclear and cytosolic fractions. Protein levels were equalized, and Diversin was precipitated using anti-FLAG beads. V5.AF9 was only detected in the nuclear precipitate. The purity of the fractions was controlled by anti-Lamin A (nucleus) and anti-α-tubulin (cytosol) staining. (B) V5-tagged wild-type Diversin or a mutant lacking the NLS sequences were coexpressed with FLAG9.AF9. Wild-type (WT), but only small amounts of mutant Diversin ΔNLS) interacted with AF9 precipitated by an antibody directed against the V5 epitope from whole cell lysates. (C) YFP.Diversin (green) and CFP.AF9 (false-colored, red) were coexpressed in HEK 293T cells. Overlapping signals were detected in the nucleus (Hoechst, blue). (D) The YFP.Diversin ΔNLS mutant (green) was retained in a perinuclear compartment, and not present in the nucleus (Hoechst, blue). YFP.Diversin ΔNLS did not co-localize with CFP.AF9 (false-colored, red). (E) 200 HEK293T
cells transfected with both Diversin and AF9 were analysed at two different time points by confocal microscopy for the presence of nuclear Diversin.
