Figure 5.

Influence of DNA binding on the dephosphorylation of Stat1, its intranuclear mobility, and the duration of nuclear accumulation. (A) Time course of Stat1 tyrosine dephosphorylation in U3A cells transiently expressing Stat1 variant proteins. Cells were prestimulated with IFNγ for 30 min to induce tyrosine phosphorylation (lane IFNγ). After removal of IFN, the cells were treated without or with staurosporine for 0.5, 2, or 4 h as indicated. Whole-cell extracts were analyzed by Western blotting with anti-P-Stat1 antibody and reprobed with anti-Stat1 antibody. (B) FRAPanalysis of the nuclear fluorescence in IFNγ-stimulated HeLa cells expressing Stat1–GFPvariant proteins. Shown is the fluorescence recovery in the bleach area in the absence (black squares) or presence (gray squares) of vanadate/H₂O₂. Note the extended time scale for Stat1dna^plus. (C) HeLa cells expressing the indicated Stat1–GFPvariant proteins were stimulated with IFNγ for 30 min to induce nuclear accumulation. Thereafter, the incubation continued in the absence of IFN for the indicated times. Shown are the GFPfluorescence and the Hoechst-stained nuclei of fixed cells.