FIGURE 4.

Interaction between CUEDC2 and SOCS3 is essential for inhibition of CUEDC2 on STAT3 activity. A, shown are deletion mutants of CUEDC2 used in domain-mapping experiments; numbers indicate amino acids included in constructs. B, purified GST or truncated GST-CUEDC2 fusion proteins were incubated with cell lysates from HEK293T cells transiently transfected with FLAG-SOCS3 vector. After extensive washes, bound proteins were analyzed by immunoblotting (IB) with anti-FLAG antibody. The GST fusion proteins were resolved by SDS-PAGE and stained with Coomassie Blue. C, shown is a schematic diagram depicting different SOCS3 deletion mutants used in the domain-mapping experiments. D, HEK 293T cells were transiently transfected with FLAG-SOCS3 or its truncated vectors, cell lysates were prepared and incubated with GST or GST-CUEDC2, and bound proteins were analyzed by immunoblotting with anti-FLAG antibody. E, HEK 293T cells were transfected with FLAG-STAT3 vectors (500 ng/well) and plasmids expressing full-length CUEDC2 or CUEDC2 deletion mutants (1.5 μg/well) and then treated with or without IFN-α (50 ng/ml) for 20 min. Lysates immunoprecipitated (IP) with anti-FLAG and whole-cell lysates were analyzed by immunoblotting with indicated antibodies. F, full-length CUEDC2 or CUEDC2 mutant expression vectors (500 ng/well) were cotransfected with pACT-Luc (200 ng/well) and FLAG-STAT3 (200 ng/well) into HEK293T cells and left treated with or without IFN-α (50 ng/ml) for 6 h, then luciferase reporter assays were performed. Data are representative of at least three independent experiments and are shown as means ± S.E.