FIG 3.

TBK1 negatively regulates NF-κB. (A) Wild-type and mutant HeLa TBK1^−/− cells were mock treated or infected with SeV for 9 h and subjected to subcellular fractionation. Nuclear extracts were prepared and subjected to EMSA using a γ-³²P-labeled κB motif DNA probe. The complex of NF-κB and probe is indicated by an arrow. For blocking assay, the extracts were incubated with the indicated antibodies for 10 min prior to probe addition (left). EMSA bands intensities were quantified (right). A representative result of three independent experiments is shown. (B) Wild-type and mutant HeLa TBK1^−/− cells were either mock treated, infected with SeV for 9 h, or treated with recombinant TNF-α (20 ng/ml) for 30 min before lysis. Whole-cell extracts were examined by immunoblotting using the indicated antibodies. (C) Wild-type and mutant HEK293T TBK1^−/− cells were transfected with reporter genes p-55A2 and pRL-TK. Where indicated, cells were cotransfected with the expression vector for TBK1. Twenty-four hours later, cells were left unstimulated (mock) or stimulated with SeV infection as indicated. Luciferase activity was quantified using pRL-TK activity as an internal reference (n = 3). (D) Wild-type and mutant HeLa TBK1^−/− cells were mock treated or infected with SeV for 9 h in the presence or absence of TPCA-1 (20 μM). The expression of the IL-8, CXCL1, and CXCL2 genes was measured by qRT-PCR (n = 3). (E) HeLa cells were mock treated or infected with SeV for 9 h in the presence or absence of BX795 (10 μM). The expression of the IL-8, CXCL1, CXCL2, and IFNB1 genes was measured by qRT-PCR (n = 3). Data are presented as means ± SEM. The Student t test was used for statistical analysis.