FIGURE 5.

CUEDC2 cooperates with and requires SOCS3 to inhibit STAT3 activation. A, FLAG-STAT3 expression vectors (500 ng/well) were transfected alone or together with expression constructs of HA-CUEDC2 (500 ng/well) and/or HA-SOCS3 (100 ng/well) into HEK293T cells. 24 h after transfection, cells were stimulated with or without IFN-α (50 ng/ml) for 20 min, lysates were immunoprecipitated (IP) with anti-FLAG, and the immunoprecipitates and whole-cell lysates were analyzed by immunoblot as indicated. B, HEK 293T cells were transfected as in Fig. 1 with HA-CUEDC2 (100 ng/well) and/or HA-SOCS3 (20 ng/well) vectors. 24 h later cells were treated with IFN-α (50 ng/ml) for 6 h, and luciferase activity was determined and normalized for transfection efficiency. Data are shown as means ± S.E. C, HEK293T cells were transfected with either control siRNA or SOCS3 siRNA 24 h before the cotransfection CUEDC2 vectors (1.5 μg/well) and FLAG-STAT3 vectors (500 ng/well). After another 24 h, cells were treated with IFN-α (50 ng/ml) for 20 min and immunoprecipitated with anti-FLAG, and whole cell lysates (WCL) were analyzed by immunoblotting with indicated antibodies. D, HEK293T cells were transfected the same as in C but not with FLAG-STAT3, and phosphorylation of endogenous STAT3 was analyzed by immunoblotting. E, HEK293T cells were transfected with control siRNA or SOCS3 siRNA 24 h before the cotransfection CUEDC2 vectors (200 ng/well) and FLAG-STAT3 vectors (200 ng/well), and luciferase activity was determined and normalized for transfection efficiency. Data are shown as the means ± S.E.