Figure 5.

Nuclear accumulation of β-catenin is insensitive to nuclear Ran-GTP depletion in living cells. A mixture of 0.8 mg/ml FLAG-β-catenin and 0.5 mg/ml GST-NLS-GFP (a and b) or 1 mg/ml GST-M9-GFP (c and d) was injected into the cytoplasm of tsBN2 cells cultured at nonpermissive temperature (upper panels). After further incubation for 30 min at nonpermissive temperature, cells were fixed, and localization of FLAG-β-catenin (b and d) was examined as in Figure 2. Localization of GST-NLS-GFP (a) or GST-M9-GFP (c) shows that nuclear accumulation of these two substrates declined in the tsBN2 cells incubated at nonpermissive temperature, whereas nuclear accumulation of FLAG-β-catenin was not affected in the same cells. Lower panels, control experiments showing that GST-NLS-GFP (a), GST-M9-GFP (b), and FLAG-β-catenin (c) all migrate normally in the nucleus of tsBN2 cells, which were incubated at permissive temperature within 30 min after cytoplasmic injection.