Figure 3

Nuclear import of fluorescein-labeled influenza vRNPs. Fluorescein-labeled influenza vRNPs are competent for nuclear import. Nuclear import assays were carried out in digitonin-treated HeLa cells, and cells were visualized by confocal microscopy. Representative images of three independent experiments are shown. (A) Control experiment with a 70 kDa dextran fluorescently-labeled with Texas Red to verify that the plasma membrane, but not the nuclear envelope, is permeabilized by digitonin. (B) Cy3-labeled BSA carrying a classical NLS (Cy3-NLS-BSA) and fluorescein-labeled influenza vRNP complexes (fluorescein-vRNP) were assayed in the digitonin permeabilized HeLa cells. Nuclear import assays were carried out in import buffer alone (- energy - cytosol) or in the presence of exogenous cytosol and an energy-regenerating system (+ energy + cytosol). (C) Bar diagram of the ratio of nuclear-to-cytoplasmic fluorescence for the experimental conditions shown in A and B. Each bar graph shows the mean value and standard error from 100–110 individual cells.