Fig. 1

A, B Cytoplasmic retention of Flag-tagged WT STAT1 in IFNγ-stimulated cells co-expressing R602L/Y701F-GFP. Transfected STAT1-negative U3A and HeLa cells expressing R602L/Y701F-GFP and WT-Flag were treated with 50 ng/ml IFNγ for 45 min and subsequently stained with an anti-Flag antibody. A The fluorescence micrographs show the intracellular distribution of Flag-tagged STAT1 co-expressed in cells transfected with R602L/Y701F-GFP as well as the localization of the corresponding Hoechst-stained nuclei (n = 3 independent transfections). B The histogram demonstrates the nucleocytoplasmic STAT1-Flag distribution in the absence and presence of R602L/Y701F-GFP in IFNγ-treated HeLa cells, as determined by the ratio of nuclear-to-total fluorescence intensity (means ± standard deviations from n = 20 cells, *p ≤ 0.05, as assessed by a two-tailed Student’s t test). C The absence of nuclear P-STAT1 staining of the endogenous protein in HeLa cells expressing R602L/Y701F-GFP. HeLa cells were transfected with R602L/Y701F-GFP and stimulated with IFNγ for indicated times before staining with an anti-phospho-tyrosine antibody. D Crystal structure of the STAT1 anti-parallel dimer. A critical arginine residue at position 274 marked in cyan in the coiled-coil domains and threonine residue at position 385 marked in pink in the DNA-binding domains of the two STAT1 proteins (white and orange). Structural data were obtained from the Protein Data Bank (pdb) file 1YVL [6] for the STAT1 anti-parallel dimer. E The absence of nuclear P-STAT1 staining of endogenous STAT1 in HeLa cells expressing R274W/R602L/Y701F-GFP. HeLa cells were transfected with R274W/R602L/Y701F-GFP and stimulated with IFNγ for indicated times before being stained with an anti-phospho-tyrosine antibody. Scale bars in A, C, E mark a distance of 10 µm