Fig. 6
A–F: The D427H mutation in STAT3 markedly reduces its dissociation from DNA but does not grossly alter transcriptional activity. A, B Electrophoretic mobility shift assay displayed the reduced dissociation of D427H complexes from radioactively labelled M67 GAS sites on DNA upon competition with a 750-fold molar excess of unlabelled M67 GAS probes. Extracts from GFP- and SNAP-STAT3 expressing IL-6-stimulated U3A cells were incubated with [33P]-radioactively labelled M67 probe for 15 min before adding an excess of unlabelled GAS probes for 10 min. Autoradiogram depicts the increased residence time of STAT3-D427H on DNA (A) and the quantification thereof from three independent transfection experiments (B). Arrows indicate the respective STAT3-GFP or STAT3-SNAP bands of interest. C, D Luciferase reporter gene assays in reconstituted U3A cells expressing the indicated GFP-tagged (C) or SNAP-tagged STAT3 variants (D) normalized to the expression level of constitutively co-expressed β-galactosidase. The reporter construct used in these experiments contained triple GAS sites of the Ly6E gene (3xLy6E) in its promoter. Cells were left untreated or stimulated for 6 h with 50 ng/ml of recombinant IFNγ or 25 ng/ml of IL-6 before luciferase luminescence and the enzymatic activity of the co-expressed β-galactosidase were measured in whole cell extracts. The experiment was repeated in six independent transfections at least three times. E, F Endogenous gene expression by the STAT3 mutants was determined by real-time PCR. Data depict expression levels of the CyclinD1 (E) and c-Myc (F) genes before and after 3 h stimulation with IL-6 or IFNγ. Gene induction was normalized to the expression of the house-keeping gene GAPDH. Histograms show means and standard deviations, wherein significant differences for comparison to the WT protein are marked by asterisks. The experiment was repeated three times in two independent transfections