Figure 4

Mutation of STAT3 at Y640 restores IFNα2-induced STAT3 transcriptional activity. (a) Left panel: HEK293T cells were transiently transfected with the pXP2d2-rPAP1-luciferase reporter together with the plasmid coding for the IL-6Rα chain and the control plasmid (Control Vector) or plasmids coding for different Etag-STAT3 mutants: STAT3Y705F; STAT3 Y640F; STAT3 Y640F Y705F; STAT3 Y657F; STAT3 Y657F Y705F. Cells were left unstimulated (NS) or stimulated for 24 hours with 10ng/ml IFNα2 or LIF or with 100ng/ml IL-6. Luciferase readout is expressed as a ratio between stimulated and unstimulated values. All results are representative of 3 independent experiments. Error bars indicate SD. *P < 0.05, **P < 0.01, ***P < 0.001; Student t-test. (b) Right panel: total cell extracts were blotted and membranes were probed with anti-STAT3, anti-Etag and anti-β-actin antibodies. Full-length blots are presented in Supplementary Fig. S12. (c) HEK293T cells were transiently transfected with the pXP2d2-rPAP1-luciferase reporter together with the control plasmid (Control Vector) or plasmids coding for different Etag-STAT3 mutants: STAT3Y705F; STAT3 Y640E. Cells were left unstimulated (NS) or stimulated for 24 hours with 10ng/ml IFNα2 or LIF. Luciferase readout is expressed as a ratio between stimulated and unstimulated values. All results are representative of 2 independent experiments. Error bars indicate SD. *P < 0.05, **P < 0.01; Student t-test. (d) Total cells extracts were blotted to test transfection efficiencies and the membranes probed with anti-STAT3, anti-Etag and anti-β-actin antibodies. Full-length blots are presented in Supplementary Fig. S13. qRT-PCR analysis representing the relative mRNA levels of genes (e) SOCS3 and (f) IFITM1. HEK293T cells were transiently transfected 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 24 hours either with 10ng/ml IFNα2 or LIF. Graphs represent the mRNA levels relative to the non-stimulated samples. All results are representative of 3 independent experiments. Error bars indicate SD. ***P < 0.001; 1-way ANOVA with Bonferroni test.