FIGURE 2.

Disruption of NS GTP-binding activity destabilizes NS in nucleoplasm. A, H1299 cells were treated with MPA for 12 h, followed by treatment with cycloheximide (CHX). Cells were harvested at the indicated time points. Equal amounts of proteins from lysates were used for Western blotting. Western blot bands were then quantified with ImageJ software and plotted on a graph. B, H1299 cells were transfected with either GFP-NS or GFP-NS^G1dm (NS(G261V/G265V)) expression plasmid. Cells were treated with cycloheximide and harvested at the indicated time points 24 h after transfection. Equal amounts of proteins from cell lysates were used for Western blotting to compare the levels of GFP-NS and GFP-NS^G1dm. Western blot bands were quantified and plotted on a graph as described above. C, disruption of GTP binding triggers NS relocalization from the nucleolus to the nucleoplasm. H1299 cells were transfected with GFP-NS or GFP-NS^G1dm. Cells were fixed in methanol and subjected to immunofluorescence staining.