Figure 3

The STAT3 Y640F mutation influences STAT3 subcellular distribution in response to IFNα2. (a) HEK293T cells were transiently transfected with empty vector (Control Vector), Etag-STAT3 wt or Etag-STAT3 Y640F mutant. Cells were starved for 16 hours and then either left unstimulated (NS) or stimulated with IFNα2 (10ng/ml) for the indicated times. Cells were fixed, stained with DAPI and anti-Etag and the localization of STAT3 was assessed by confocal analysis. Immunofluorescence of representative cell fields is shown. Enhanced IFNα2-induced DNA binding of the STAT3 Y640F mutant. (b) ChIP assays were performed to examine the occupancy of STAT3 on the SOCS3 promoter. HEK293T cells were transiently transfected either with empty vector (Control Vector) or different Etag-STAT3 mutants: STAT3Y705F; STAT3 Y640F; STAT3 Y640F/Y705F. Cells were serum starved 4 hours and then left unstimulated (NS) or stimulated for 1 hour either with 10ng/ml IFNα2 or LIF. Immunoprecipitated DNA was used for qRT-PCR using specific primers for the SOCS3 promoter. Graphs represent occupancy levels relative to irrelevant IgG immunoprecipitated DNA samples. All results are representative of 3 independent experiments. Error bars indicate SD. **P < 0.01, ***P < 0.001; 1-way ANOVA with Bonferroni test.