Functional properties of mutated STAT6 proteins in HEK transfectant cells. (A) Transfected HEK293 cells expressing WT or N417/N430 DM STAT6, selected with G418 (transfection 1, pools; 2, single clones), were incubated (+) or not (−) with 10 ng/mL IL-4 for 24 hours. Western blot analyses of total protein extracts (7 μg) probed with antibodies against P-STAT6 (Tyr-641), total STAT6, and β-actin are shown. (B) EMSA was prepared using 1 μg nuclear extracts from mock transfected cells, WT or DM STAT6 HEK293 pools, stimulated with 10 ng/mL IL-4 for 6 hours, and radiolabeled N3-GAS or N4-GAS probes. Incubation was performed with (+) or without (−) an antibody against STAT6 or an excess of cold probe. The protein/DNA complexes were electrophoresed on the same gel and are assembled side by side in the figure for better comparison. The position of the STAT6 containing complexes and of the complexes supershifted (SS) by the antibody are indicated. Phosphorimager quantification of the radiolabeled complexes is shown in the left plots, and Western blot analysis of P-STAT6 and HDAC, used as a loading control, in nuclear extracts is shown in the right plot. (C) HEK293 cells were cotransfected with expression vectors for WT STAT6, single or DM STAT6 mutants, or a control mock-vector, together with 3 × N3-luc (left plot) or with 3 × N4-luc (right plot) luciferase reporter constructs. The diagrams show the mean percentage of luciferase activity, after normalization for transfection efficacy, in cells treated () or not () with 10 ng/mL IL-4 for 20 hours, compared with WT STAT6 expressing cells (set to 100%). Error bars shows the SD (3 independent experiments). The amount of P-STAT6 in total protein extracts of IL-4-treated cells, detected by immunoblotting, is shown in the bottom panel.