Fig. 3: Stub1 targets TBK1.
A HEK293T cells were transfected with an IFN-β luciferase reporter and a Renilla-TK reporter and a plasmid expressing RIG-I, MDA-5, MAVS, TBK1, IKKε, IRF3-5D, or cGAS+STING, along with an empty vector or a plasmid expressing Flag-Stub1. The Luc activity of the IFN-β promoter-reporter was detected. B HEK293T-stub1+/+ and HEK293T-stub1−/− cells were transfected with an IFN-β luciferase reporter, a Renilla-TK reporter, and a plasmid expressing RIG-I, MDA-5, MAVS, TBK1, IKKε, IRF3-5D, or cGAS+STING. The luciferase activity of the IFN-β promoter reporter was detected. C HEK293T cells were transfected with plasmids expressing Myc-Stub1 alone or together with plasmids expressing Flag-RIG-I, MDA5, MAVS, TBK1, IKKε, IRF3, cGAS, or STING for 24 h. The cell lysates were harvested and used for Co-IP. Input and IP complexes were analyzed by Western blotting using antibodies against Flag and Myc. D HEK293T cells were transfected with plasmids expressing HA-Stub1 alone or together with a plasmid expressing Flag-TBK1. At 24 hpt, the cell lysates were harvested and used for reverse Co-IP. Input and IP complexes were analyzed by Western blotting using antibodies against Flag and HA. E His-TBK1 was incubated with GST or GST-Stub1 for 30 min, and the protein-protein interactions were then confirmed via a GST pull-down assay. F Bimolecular fluorescence complementation (BIFC) analysis of TBK1 and Stub1. HA-Stub1-VN, Stub1 fused with the N-terminus of Venus; Flag-TBK1-VC, TBK1 fused with the C-terminus of Venus. G, H Co-IP and immunoblot analysis of the interaction of endogenous Stub1 and TBK1 in THP-1 cells infected with VSV (0.1 MOI) (F) or HSV-1 (10 MOI) (G) for 12 h. Co-IP was subsequently performed with an anti-stub1 antibody. IgG was used as a negative control. I THP-1 cells were mock-infected or infected with VSV (0.1 MOI) or HSV-1 (10 MOI) for 12 h. The subcellular localization of endogenous Stub1 and TBK1 was analyzed via fluorescence microscopy. Ns not significant (P > 0.05), *0.01 < P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed Student’s t test). The data represent three independent experiments with three biological replicates (means ± SDs) or three independent experiments with similar results (A–I).
