Knockdown of SOCS3 activates the IFN-β signaling pathway. (A) qRT-PCR (left) and RT-PCR (right) analysis of SOCS3 knockdown efficiency at the mRNA level in HeLa cells. (B) qRT-PCR analysis of SOCS3 knockdown efficiency at the mRNA level in 293T cells. (C) Immunoblot analysis of the knockdown of exogenous SOCS3 in 293T cells transfected with Flag-tagged SOCS3 plasmid and siRNA control or SOCS3-specific siRNAs. (D) Real-time PCR analysis of IFN-β mRNA in 293T cells transfected with control siRNA or siRNA against SOCS3. (E) 293T cells were transfected with siRNA control (si-control) or si-1 for 36 h and further transfected with plasmids harboring genes for the IFN-β promoter and TK reporter genes, with or without a TBK1 expression vector. The activity of the IFN-β promoter was analyzed in dual-luciferase assays. (F) HeLa cells were transfected with si-control or si-1 and further transfected with or without TBK1 plasmid. The transcription of IFN-β mRNA was evaluated by real-time PCR. (G) Thirty-six hours after transfection with siRNAs, 293T cells were transfected with the indicated plasmids. Luciferase assays were performed to evaluate the activity of the ISRE reporter. (H) Immunoblot analysis of IRF3 phosphorylation in 293T cells transfected with IRF3 plasmid (50 ng/ml) and si-control or si-1. (I) Immunoblot analysis of IRF3 phosphorylation in 293T cells transfected with the indicated siRNA or SOCS3 rescue plasmid. Overexpressed and endogenous SOCS3 is indicated with an arrow. (J) Primary MEF cells were transfected with si-control or si-1. The expression levels of TBK1 and SOCS3 were evaluated by IB analysis. Error bars indicate standard deviations. *, P
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