Figure 8. Selective response of FrAdV1 Pre-pVII NLS substitution derivatives to nuclear transport inhibitors unveils the specific interaction of individual NLSs with cellular IMPs. (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 letteramino 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 merged images of 633 nm (nuclei, blue ), 488 nm (GFP, green ), and 561 nm (inhibitors, red)laser lines are shown. Images of individual channels and additional histograms are shown in the Supplementary Figs S3-S10. (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 20 cells from three independent experiments. Results from Welch and Brown-Forsythe ANOVA test for accumulation of the indicated GFP fusion protein expressed in the absence or in the presence of the indicated nuclear transport inhibitors. *:p < 0.05; **:p < 0.01; ***: p < 0.001; ****: p < 0.0001; ns: non-significant.