TBK1 promotes STING aggregation.

<p>(A) TBK1 promoted STING aggregation in a dose-dependent manner. HEK293 cells were transfected with STING-Myc expression plasmids (0.2 μg) together with the empty vector (2 μg) or increased dose of Flag–TBK1 expression plasmids (0.5 μg, 2 μg). At 30 h after transfection, the cell lysates were resolved by SDS-PAGE (top), native gel electrophoresis (middle), or SDD-AGE (bottom), and analyzed by immunoblotting with the indicated antibodies. (B) TBK1 induced STING phosphorylation. HEK293 cells were transfected with STING-Myc (0.5 μg) along with empty vector (2 μg) or Flag-TBK1 (2 μg), and 24 h later, cell lysates were immunoprecipitated with anti-Myc. The immunoprecipitates were treated with buffer or calf intestine phosphatase (CIAP) and analyzed by with SDS-PAGE, followed by immunoblotting. (C) DTT treatment reduced STING aggregation. HEK293 cells were transfected with STING-Myc expression plasmids together with the empty vector or Flag–TBK1 expression plasmid. At 30 h after transfection, the cell lysates were untreated or treated with 5 mM or 20 mM DTT for 30 min at room temperature, and then resolved by SDD-AGE (upper panel) or SDS-PAGE (lower panels), followed by immunoblotting analysis with the indicated antibodies. (D) CIAP treatment reduced the STING aggregation induced by TBK1. HEK293 cells were transfected with STING-Myc expression plasmids along with empty vector or Flag–TBK1 expression plasmids. At 24 h after transfection, the cells were lysed and then left untreated or treated with CIAP for 30 min at 37°C as indicated and analyzed with SDD-AGE and SDS-PAGE, followed by immunoblotting analysis. (E) TBK1 kinase activity was important for the induction of STING aggregation. HEK293 cells were transfected with the indicated plasmids for 24 h. The cell lysates were separated with SDD-AGE and SDS-PAGE, and analyzed by immunoblotting with the indicated antibodies. (F) Endogenous STING formed aggregates in response to stimulation with ISD in THP-1 cells. THP-1 cells were transfected with ISD (2 μg/ml) using Lipofectamine 2000 for the indicated times and lysed. The proteins were resolved with SDD-AGE and SDS-PAGE, and analyzed by immunoblotting with the indicated antibodies. (G) Knockdown of endogenous TBK1 attenuated STING aggregation in HEK293 cells. HEK293 cells were transfected with NC (40 nM) or a TBK1 (40 nM) RNAi oligonucleotide. At 48 h after transfection, the cells were transfected with vector encoding STING-Myc or empty vector for 24 h. The cell lysates were prepared and resolved with SDD-AGE (upper panel) and SDS-PAGE assays (lower panel), followed by immunoblotting with the indicated antibodies.