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.