Fig. 6

STAT1-E169A binds to DNA with high affinity. HeLa or U3A cells expressing fusion proteins of WT or mutant STAT1 were left unstimulated or stimulated with 50 ng/ml IFNγ for 45 min, and DNA-binding properties were assessed using different DNA probes containing GAS motifs. A Representative autoradiogram from HeLa whole cell extracts. HeLa cells were treated with IFNγ for 45 min and subsequently with staurosporine for the indicated times, and whole cell extracts were incubated with M67 probe for 5 min on ice before undergoing electrophoresis. The arrow indicates bands of STAT1-DNA complexes, and the asterisk marks an unspecific band. B Quantification of EMSA results from three independent transfection experiments as shown in (A). C, D Representative autoradiogram from U3A whole cell extracts expressing the indicated untagged STAT1 proteins (C), including quantification of results thereof from three independent transfection experiments (D). E Representative autoradiogram from U3A whole cell extracts. Samples were incubated for 5 min on ice prior to electrophoresis with DNA probes containing either no (2 × non-GAS), one (GAS-nonGAS) or two GAS sites (2 × GAS), as indicated. The asterisk marks a non-specific band. F Quantification of EMSA results from three independent transfection experiments as shown in (E). G Representative autoradiogram from U3A whole cell extracts using a competition EMSA experiment. Samples were incubated with the labeled M67 probe on ice for 30 min and then challenged with a 750-fold excess of unlabeled M67 probe for the indicated times. The asterisk marks a non-specific band. H Quantification of EMSA results from three independent transfection experiments as shown in (G). In all graphs, asterisks indicate significant differences between the WT protein and the respective mutant