Extended Data Fig. 5: Binding studies of human STING with human TBK1 mutants and characterization of TBK1-knockout HEK293T cells.
From: A conserved PLPLRT/SD motif of STING mediates the recruitment and activation of TBK1
a–g, SPR binding studies of the phosphorylated human STING CTD (residues 155–379) with human TBK1 mutants in the presence of 1 μM cGAMP. The binding affinity (Kd) was determined by fitting the binding data to a one-site binding model. SDS–PAGE analysis of proteins used in these studies is shown in the inset of panel a. h, Western blot characterization of TBK1-knockout HEK293T cell lines. i, IFNβ luciferase reporter assays using TBK1-knockout cells. For each assay, 0.2 ng pcDNA3.1-hSTING plasmids and/or 1.0 ng pcDNA3.1-hTBK1 plasmids were transfected into TBK1-knockout cells. Data are mean ± s.e.m. and representative of three independent experiments. Each dot represents a technical replicate (n = 3). ∗∗∗P < 0.001, two-tailed Student’s t-test. j, Western blot showing the phosphorylation of TBK1, STING and IRF-3 in TBK1-knockout cells transfected with STING and TBK1 plasmids. TBK1-knockout cells were transfected with 0.2 ng pcDNA3.1-hSTING plasmids and/or 1.0 ng pcDNA3.1-hTBK1 plasmids and stimulated with cGAMP. k, Immunoprecipitation and immunoblot of Flag–STING and TBK1 in TBK1-knockout cells. The cells were transfected with Flag–STING and TBK1 mutants and stimulated with cGAMP. Flag–STING and TBK1 in the pull-downs and whole-cell lysates were analysed by immunoblotting. STING was visualized with Flag antibody. TBK1 in the pull-downs was detected with an antibody against TBK1. Data in a–h are representative of at least two independent experiments. Western blot data in j and k are representative of three independent experiments.
