Fig. 2

The dimerization-deficient quadruple mutant (QM-GFP) mimicking U-STAT1 inhibits the detection of co-expressed WT protein but does not affect tyrosine phosphorylation. A–C HeLa cells were transfected with a plasmid coding for QM-GFP and stimulated with IFNγ for 45 min followed by staining with anti-phospho-tyrosine antibody. A The fluorescence micrographs show the intracellular distribution of QM-GFP and WT P-STAT1, as well as the localization of the corresponding Hoechst-stained nuclei. B Histogram demonstrating the net reduction of nuclear P-STAT1 intensity in HeLa cells expressing QM-GFP, as determined by the ratio of nuclear-to-total fluorescence intensity (n = 3 independent transfections, means ± standard deviations from n = 20 cells, *p ≤ 0.05 by a two-tailed Student’s t test). C Concentration-dependent inhibition of nuclear P-STAT1 accumulation by co-expressed QM-GFP. The fluorescence intensities of nuclear P-STAT1 staining were plotted against the total cellular QM-GFP fluorescence. D, E Representative immunoblot of whole cell extracts from STAT1-negative U3A cells co-expressing untagged STAT1 and QM-GFP after treatment for 45 min with 50 ng/ml of recombinant IFNγ and the quantification thereof from three independent transfection experiments. F, G An in vitro phosphorylation assay demonstrated no difference in tyrosine phosphorylation rates of the WT STAT1 by JAK2 with respect to the presence or absence of QM-GFP. Whole cell extracts (10 μl in each reaction) from reconstituted U3A cells expressing untagged STAT1 in combination with WT-GFP or QM-GFP were incubated with 4 μg/ml of recombinant JAK2 kinase and the levels of P-STAT1 were monitored over time by means of Western blotting (n = 3). Statistical analysis revealed no significant difference in the phosphorylation kinetics of the WT in the presence of either QM-GFP or WT-GFP. H, I Results from an in vitro dephosphorylation assay using extracts from IFNγ-pre-stimulated U3A cells expressing untagged STAT1 in combination with WT-GFP or QM-GFP (10 μl each) incubated for 0, 15 and 30 min with 2 U of the STAT-specific Tc45 phosphatase (n = 3). Tyrosine dephosphorylation was followed by immunoblotting including a quantitative analysis of the phospho-tyrosine signals divided by total amount of STAT1 signal. Scale bar in A marks a distance of 10 µm