FIG 2.

Effect of multiple amino acid substitutions on the NLS in H-IPSE. (A) The nucleotides encoding the H03/H06-IPSE nuclear localization sequence (SKRRRKY and SKRGRKY, respectively) were inserted into the pTetra-EGFP construct (2, 3). pTetra-EGFP encodes a tetrameric EGFP construct resulting in the expression of a fluorescent protein, which, due to its size (>100 kDa), is excluded from the nucleus in the absence of a functional NLS (tetra-EGFP) or imported into the nucleus in the presence of a functional NLS (canonical SV40 NLS and H03/H06-IPSE NLS). Nuclei were stained with DAPI, and green fluorescence was measured with the GFP light cube on an Evos fl microscope, 24 h after transfection. Bar, 100 μm. (B) Comparison of the effects of wild-type H06-IPSE and H03-IPSE and mutant H03-IPSE NLSs on the nuclear localization of the tetra-EGFP fusion protein. One hundred transfected HTB9 cells were evaluated under an Evos fl microscope for each transfection, and the percentage of cells displaying exclusively nuclear fluorescence, as opposed to cytosolic localization only or mixed cytosolic/nuclear localization, was recorded. The positive control was the SV40 canonical NLS, and the negative control was the unmodified tetra-EGFP vector (Tetra-EGFP).