Fig. 3: TBK1-dependent phosphorylation controls the binding of STING to AP-1.
From: Clathrin-associated AP-1 controls termination of STING signalling
a, Schematic diagram of the CTT of human STING (hSTING) and sequence logo of the CTT as indicated from 50 species. b, HeLa STING-knockout (KO) cells transfected with Flag-tagged STINGΔCΤΤ (Δ1–341), wild-type (WT) STING, STINGE(E360A), STINGLI(L364A/I365A) or STINGELI(E360A/L364A/I365A) were treated with diABZI for 2 h. After immunoprecipitation with anti-Flag antibody, samples were analysed by western blot. c, WCL and extracted CCV fractions from HeLa STING KO cells reconstituted with Flag-tagged wild-type STING or STINGLI(L364A/I365A) and treated or not with diABZI were analysed by western blot. CHC was used as a loading control. d, Glutathione sepharose pull-down assays of wild-type LBD-STING or LBD-STINGELI by glutathione S-transferase (GST)-tagged AP-1 core with or without ARF1. e, HeLaSTING cells transfected with NC siRNA or siRNAs against TBK1 or IRF3 were treated with or without diABZI. After immunoprecipitation with anti-Flag antibody, samples were analysed by western blot. GAPDH was used as a loading control. f, HeLa wild-type cells, HeLa TBK1 KO cells and HeLa IRF3 KO cells stimulated with diABZI for 0, 1, 2 or 4 h were analysed by western blot. Ratios of target proteins versus loading control normalized to the 0-h time point of each condition. Vinculin was used as a loading control. g, Bio-layer interferometry binding studies of LBD-STING (top) or TBK1-phosphorylated LBD-STING (pSTING) (bottom) with AP-1 ΔμCTD. The right graphs show the binding affinity of STING (top) and pSTING (bottom). One representative example of at least three (b,d,f) or two (c,e,g) independent experiments is shown.
