Figure 7. Dissection of the individual contribution of each
putative NLS to FrAdV1 nuclear and nucleolar targeting. (A) Schematic
representation of the GFP-FrAdV1 Pre-pVII fusion proteins expressed.
Proteins are represented by a white horizontal bar . NLSs are
shown as blue vertical bars , along with their amino acidic
sequence. Individual amino acidic substitutions are indicated by arrows
and their sequence is in red . The single letter amino acid
code is used. Basic residues are in boldface . (B) The indicated
GFP fusion proteins were transiently co-expressed with DsRed-fibrillarin
in HEK293A by means of Lipofectamine 2000 transfection. 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 of the 633
nm (nuclei), 488 nm (Pre-pVII), and 568 (fibrillarin) laser channels are
shown, along with a merged image (merge), and a rgb profile plot across
the indicated area. (C, D) Micrographs such as those shown in (B) were
quantitatively analysed to calculate the levels of nuclear (C) or
nucleolar (D) accumulation 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
are means (black horizontal bars) + standard error of the mean
(SEM), relative to pooled data from at least 70 cells from three
independent experiments. Results from Welch and Brown-Forsythe ANOVA
test for accumulation of the indicated mutant Pre-pVII proteins as
compared to the wild-type protein. ***: p < 0.001; ****: p
< 0.0001.