Fig. 4

Deletion of the amino-terminus in mutant U-STAT1 restores nuclear accumulation of co-expressed, endogenous P-STAT1. A, B HeLa cells were transfected with an expression plasmid coding for R602L/Y701F-GFP, QM-GFP, ΔN/R602L/Y701F-GFP and QM/L407A/L409A-GFP and stimulated for 0 min or 45 min with 50 ng/ml of recombinant IFNγ, followed by staining with an anti-phospho-tyrosine antibody. Fluorescence micrographs show restored nuclear accumulation of P-STAT1 in the presence of amino-terminal deletion mutant ΔN/R602L/Y701F-GFP and the dsNLS mutant QM/L407A/L409A-GFP. Histograms show the quantification of nuclear P-STAT1 from HeLa cells co-expressing the indicated STAT1 variants in comparison with untransfected HeLa cells from three independent experiments (n = 3, means ± standard deviations from n = 20 cells, *p ≤ 0.05). C Fluorescence micrographs displaying the absence of nuclear endogenous P-STAT1 staining in the presence of mutant U-STAT1 constructs expressed in HeLa cells stimulated for 0 min and 45 min with recombinant IFNα (50 ng/ml). Note the rescue effect mediated by the deletion of the amino-terminus in the construct ΔN/R602L/Y701F-GFP under stimulation with type I IFN. Scale bars in A, C mark a distance of 10 µm. D Cellular fractionation experiments using cytoplasmic and nuclear lysates from HeLa cells expressing the indicated STAT1 variants demonstrate the presence of endogenous phospho-STAT1 in both compartments in IFNγ-pretreated cells. Representative immunoblots demonstrate unchanged tyrosine phosphorylation of endogenous STAT3 in whole cell extracts from HeLa cells expressing U-STAT1, after stimulation with IFNγ (lower panels). E, F Quantification from immunoblots of whole extracts from HeLa cells expressing the indicated mutants, including the quadruple mutant (QM), before and after treatment for 45 min with 50 ng/ml of recombinant IFNγ from three independent transfection experiments for the expression of phospho-STAT1 (E) and the expression of phospho-STAT3 (F)