FIGURE 4.

Nuclear accumulation of the NC2 complex via importin α/β requires the cNLSs of both subunits. A, HeLa P4 cells were transiently co-transfected with plasmid DNA encoding wild-type (wt) and mutated RFP-NC2α and EGFP-NC2β, respectively. The subcellular distribution of the gene products was examined 24 h post-transfection by direct fluorescence. The DNA was counterstained with Hoechst. Co-expression of wild-type RFP-NC2α and EGFP-NC2β results in a nuclear co-localization shown in yellow (merge; top panel). Mutation of either the cNLS of NC2α (K5A) or the cNLS of NC2β (R101A) reduced the nuclear accumulation leading to a homogeneous localization of both subunits (middle panels). Nuclear import of the RFP-NC2α/EGFP-NC2β complex was strongly blocked when the cNLSs of both subunits were mutated (bottom panel). B, for quantitative analysis, the mean red (RFP) and green (EGFP) fluorescence value in the nucleus and cytoplasm of 25 cells that co-expressed RFP-NC2α and EGFP-NC2β was measured using the ImageJ Software (NIH). After subtraction of the background value the percentage of nuclear localization of the different NC2 complexes was calculated. Bars indicate the mean ± S.D.