Basic amino acids required for targeting GFP into nucleus.

<p>(A) The amino acid sequence covering residue 21 to 63 defined as CT311 fragment 4 (F4) or nuclear localization signal sequence (NLS) was shown with two clusters of basic residues highlighted in red and marked as Clusters 1 & 2 (C1 & C2) respectively. Cluster 2 consists of 2 separate basic residue sub-clusters, designated as C2a & C2b respectively. The sequences of constructs with C2b deletion (C2b del-GFP) and C1 or C2a substitution mutations (C1 sub-GFP or C2a sub-GFP) were also listed. (B) HeLa cells transfected with pLEGFP plasmid alone or expressing the various constructs listed in (A) were processed and observed under a fluorescence microscope as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064529#pone-0064529-g004" target="_blank">Fig. 4B</a> legend. Deletion or substitution of C2b, C1 or C2a effectively blocked the nuclear targeting of GFP by the CT311 NLS. (C) The GFP signal in the nuclei and entire cells was semi-quantitatively measured and the % of nuclear GFP signal from each sample was compared between cell samples as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064529#pone-0064529-g004" target="_blank">Fig4</a>. ** indicates statistically significant differences (P<0.01) and “ns” stands for no significant difference. The data were from 3 independent experiments.