Fig. 5. Altered localization of an IN DNA-binding mutant. (A) Wild-type IN (a-c) or the DNA-binding defective mutant K156E/K159E (d-f) was localized in transiently transfected HeLa cells following incubation in DMSO (a,d) or 5 µM MG-132 for 5 hours (b,c,e,f). Notice that the DMSO-treated cells expressing K156E/K159E IN exhibited a considerable degree of cytoplasmic staining (d), a pattern that mirrored wild-type IN stabilized with MG-132 (b,c). Following MG-132 treatment, cells expressing K156E/K159E IN exhibited essentially diffuse nuclear and cytosolic staining (e,f), although some residual nuclear accumulation was also observed (e,f, arrows). (B) Quantitation of nuclear and cytoplasmic distribution of IN and K156E/K159E IN. Indirect immunofluorescence microscopy was performed and ten fields of view were captured for each transfection condition (total of 200 transfected cells). The intensity of nuclear IN (based on colocalization with DAPI) versus cytoplasmic IN (signal outside of DAPI) was quantified for each field of view using MetaMorph software. For each condition, the mean percentage of cytoplasmic IN signal across ten fields of view is presented. Error bars represent standard deviation between the ten fields of view. (C) HeLa cells stably expressing IN or K156E/K159E were examined by indirect immunofluorescence microscopy. K156E/K159E-IN-expressing cells frequently exhibited more cytoplasmic staining than WT IN cells, although significant nuclear accumulation was also observed.

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