Figure 2.

Stat1 nuclear accumulation is a dynamic process driven by nucleocytoplasmic shuttling and antagonistic kinase and phosphatase activities. (A) Time course of Stat1 tyrosine phosphorylation in HeLa cells. Cells were stimulated with IFNγ for 30 min to induce tyrosine phosphorylation, at which point IFNγ was withdrawn. The incubation was continued for the indicated times without or with staurosporine (upper panel) or vanadate/H₂O₂ (lower panel), respectively. Whole-cell extracts were analyzed by Western blotting with anti-P-Stat1 antibody and reprobed with anti-Stat1 antibody. (B) Time course of accumulation of Stat1 in the nuclei of HeLa cells after 30 min of IFNγ stimulation. Treatment of cells was identical to that in A. At the indicated times, Stat1 was detected in fixed cells by indirect immunofluorescence microscopy using an anti-Stat1 antibody. (C, first and second rows) HeLa cells were stimulated with IFNγ for 30 min before anti-Stat1 antibodies and FITC-labeled BSA were comicroinjected into the cytosol. Subsequently, the incubation was continued for another 30 min with IFNγ alone (first row), or for 90 min in the additional presence of vanadate/H₂O₂ (second row). Shown are representative cells and the corresponding bar diagram with a quantitative analysis of the ratio of cytoplasmic and nuclear Stat1 immunofluorescence density for noninjected cells (control) and for injected cells (n = 12 each). Statistically significant differences are indicated by *. (Third row) Purified Stat1 Tyr701F was coinjected with FITC-BSA in the nucleus of HeLa cells. At the time point of microinjection, the cells had been pretreated with IFNγ for 60 min with vanadate/H₂O₂ present after 30 min. The microinjected cells were incubated with IFNγ/vanadate for another 45 min before fixation and immunocytochemistry. (Fourth row) Stat1 export signal fused to GFP-GST (NES-GFP) was coinjected with TRITC-BSA in the nucleus of cells that were pretreated with IFNγ/vanadate as before. After microinjection, the incubation was continued for another 30 min in the presence of IFNγ/vanadate before the fusion protein was located in fixed cells by its GFPautofluorescence.