(A, B) The E559A and E563A mutants display high-affinity DNA binding and restore defective nuclear accumulation of a STAT1 variant with artificially enhanced nuclear export.

<p>HeLa cells transfected with pSTAT1-NES-GFP, which coded for a transferable nuclear export signal (NES) situated between the cDNAs for full-length STAT1 and GFP, were either left untreated or treated for 45 min with IFNγ (5 ng/ml), the CRM1 export inhibitor leptomycin B (LMB, 10 ng/ml) or a combination of the two. (A) The micrographs demonstrate the intracellular distribution of STAT1-NES-GFP and the localization of corresponding Hoechst-stained nuclei (scale bar 10 µm). (B) Histograms demonstrate the percentage of nuclear-to-total STAT1 fluorescence intensities for the various STAT1-NES proteins and stimulation modes. (C) <i>In vitro</i> dephosphorylation assays showing that DNA-bound E559A, E563A and K567A are protected from Tc45-catalyzed inactivation. Whole cell extracts from reconstituted U3A cells expressing wild-type or mutant STAT1 were incubated for 30 min with or without the recombinant Tc45 phosphatase in the absence or presence of 2xGAS, GAS-nonGAS or 2xnonGAS, as indicated. Reactions were probed for phospho-STAT1 levels by means of Western blotting (αSTAT1-P). The membrane was then stripped and re-incubated with the pan-STAT1 antibody C-24 (αSTAT1). (D) Precipitation of STAT1-WT, -E559A and -K567A with agarose-bound duplex oligonucleotides comprising tandem M67 binding sites (2xGAS), a GAS and non-GAS site (GAS-nonGAS) or a mutated sequence thereof (2xnonGAS). Beads were incubated with cell extracts from transfected U3A cells with and without IFNγ treatment. Five percent (vol/vol) of the cell extract input was blotted and probed with anti-STAT1 antibody. The precipitate (pull down) was analyzed by Western blotting with anti-Stat1 antibody. (E) Quantification of three independent pull-down experiments for each of the STAT1 variants tested, as shown in D. Significant differences between the three variants are indicated with an asterisk.