Figure 6. FrAdV1 Pre-pVII localize to the cell nucleus independently of IMPα/β and IMPβ2. (A) Schematic representation of the GFP-fusions and of nuclear import inhibitors used in the assay. Proteins are represented by white horizontal bars. NLSs are shown as blue vertical bars, along with their amino acidic sequence and position. The single letter amino acid code is used. Basic residues are in boldface. (B) The indicated GFP fusion proteins were transiently expressed in HEK293A by means of Lipofectamine 2000 transfection, in the absence (no add ) or in the presence of plasmids mediating the expression of either mCherry-Bimax2 (+ Bimax2 ), RFP-M9M (+ M9M ) or DsRed-RanQ69L (+ RanQ69L ). 24h post transfection, cells were incubated with DRAQ5 to stain cell nuclei, fixed with paraformaldehyde and processed for CLSM analysis as described in the Materials and Methods section. Representative images the 633 nm (nuclei ), the 488 nm (GFP ), and 561 nm (inhibitor ) channels are shown, along with a merged image (merge ). (C) Micrographs such as those shown in (B) were quantitatively analysed to calculate the levels of nuclear accumulation (Fn/c) relative to the indicated fusion proteins at the single cell level, as described in the Materials and Methods section. Data shown are individual measurements (circles ), along with means (black horizontal bars) + standard error of the mean (SEM), relative to pooled data from at least 27 cells from three independent experiments. The dashed horizontal line indicates a Fn/c of 1, which corresponds to equal distribution between nucleus and cytoplasm. Results from two-way ANOVA test for the nuclear accumulation of the indicated GFP-fusion proteins in the absence or in the presence of the indicated nuclear transport inhibitors are shown. ***: p < 0.005; ****: p < 0.0001.