Figure 2. Functional properties of mutated STAT6 proteins in HEK transfectant cells.

a) Transfected HEK293 cells expressing WT or N417/N430 double mutant (DM) STAT6, selected with G418 (transfection 1: pools, 2: single clones), were incubated (+) or not (−) with 10 ng/ml interleukin 4 for 24h. Western blots of total protein extracts (7μg) probed with antibodies against P-STAT6 (Tyr-641), total STAT6, and β-actin are shown.

b) Electrophoretic Mobility Shift Assay was prepared using 1μg nuclear extracts from mock transfected cells, WT or DM STAT6 HEK293 pools, stimulated with 10 ng/ml interleukin 4 for 6 h, 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 radiolabelled 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 co-transfected with expression vectors for WT STAT6, single or DM STAT6 mutants or a control mock-vector, together with 3 x N3-luc (left plot) or with 3 x N4- luc (right plot) luciferase reporter constructs. The diagrams show the mean percentage of luciferase activity, after normalization for transfection efficacy, in cells treated (grey bars) or not (white bars) with 10 ng/ml interleukin 4 for 20 h, compared to WT STAT6 expressing cells (set to 100%). Error bars shows the standard deviation (3 independent experiments). The amount of P-STAT6 in total protein extracts of interleukin 4 treated cells, detected by immunoblotting, is shown in the lower panel.