Fig. 5
A–E: Increased DNA affinity of the D427H mutant. Electrophoretic mobility shift assays (EMSA) were performed using lysates from IL-6- and IFNγ-stimulated U3A cells expressing STAT3-GFP/-SNAP variants incubated with a [33P]-radioactively labelled DNA probe containing a consensus GAS sequence. Autoradiograms displaying the increased DNA-binding of the D427H-GFP mutant (A, B) and D427H-SNAP mutant (C, D) upon cytokine stimulation to a single GAS element (M67) as compared to the WT protein and similar to the positive control F174A mutant, including the quantification thereof from three independent transfection experiments. E Gelshift demonstrating the reduction in sequence-specificity for the STAT3-D427H mutant. Whole cell extracts from IFNγ-stimulated U3A cells transfected with GFP- or SNAP-tagged STAT3 variants were incubated with radioactively labelled DNA probes comprising of two complete GAS sites in tandem orientation (2xGAS), one-and-a half GAS site (GAS-nonGAS), or none GAS elements (2xnonGAS). DNA-bound STAT3 was separated by electrophoresis and visualized in the autoradiogram. Means ± standard deviations from three independent experiments with *p ≤ 0.05. Arrows indicate the respective STAT3-GFP or STAT3-SNAP bands of interest