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. 2000 Feb;74(4):1892–1899. doi: 10.1128/jvi.74.4.1892-1899.2000

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Copyright © 2000, American Society for Microbiology

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FIG. 3 — NES of Int6. (A) The NES sequence of Int6, containing amino acids 1 to 20 of Int6, is shown. The leucines which are critical for the function of the NES are underlined. (B) The wt NES of Int6 was fused in frame to the N terminus of the Flag-tagged nuclear protein DRBP76 to produce NES-DRBP76-Flag and DRBP76-Flag or NES-DRBP76-Flag was transfected into HT1080 cells. Twenty-four hours posttransfection cells were fixed and immunofluorescence was performed using Flag antibody to detect the subcellular location of DRBP76-Flag or NES-DRBP76-Flag fusion proteins. Phase-contrast (left side) and immunofluorescence (right side) images are shown. The leucine at position 6, 14, or 20 of the NES of Int6 was mutated singly to alanine, and the mutated NES was fused in frame to DRBP76-Flag to produce L6ANES-DRBP76-Flag, L14ANES-DRBP76-Flag, or L18ANES-DRBP76-Flag. Then the three mutant proteins were expressed singly in HT1080 cells in the same way as described above. Immunofluorescence was performed using Flag antibody to detect the subcellular location of different mutant proteins.