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.