Fig. 3. Localization of IN fused to a strong NES. (A) Schematic of IN intracellular transport and colocalization with DNA. (B) Predicted intracellular dynamics of IN-NES. Following nuclear entry, IN-NES should be exported back into the cytoplasm. (C) HeLa cells were transiently transfected or stably infected with viruses expressing IN or IN-NES. In contrast to IN (a,b), IN-NES was localized to the cytoplasm of most transiently transfected cells (d,e). In rare instances, IN-NES was also observed within the nucleus of transfected cells (e, arrow). However, IN-NES was observed almost exclusively in the nucleus of stably expressing cells (f), a pattern indistinguishable from IN (c). (D) Western-blot analysis of untransfected cells (Unt) or cells transiently transfected or stably expressing IN or IN-NES. 10 µg of total protein was resolved by SDS-PAGE. IN and IN-NES were detected by probing blots with 12CA5 anti-HA antibody. The migration positions of molecular mass markers in kDa are indicated to the right of the gel. Notice the ten-amino-acid NES insertion, which resulted in a slight shift in migration compared with wild-type IN. *, cross-reacting band.