Extended Data Fig. 6: cGAMP binding induces the oligomerization of full-length STING.
From: A conserved PLPLRT/SD motif of STING mediates the recruitment and activation of TBK1
a, Gel-filtration chromatography analyses of full-length STING in the presence and absence of 1 μM cGAMP using a Superose 6 column. b, SDS–PAGE analyses of fractions containing full-length STING from gel filtration chromatography. c, Gel filtration chromatography analyses of full-length STING in 0.1% DDM or Amphipol A8-35 using a Superose 6 column in the presence of 1 μM cGAMP. d, SDS–PAGE analysis of cross-linked full-length STING. e, SEC-MALS analysis of full-length STING in 0.1% DDM and 1 μM cGAMP. f, Representative cryo-EM micrograph of full-length STING stabilized with Amphipol A8-35. g, Representative 2D averages of full-length STING particles in Amphipol A8-35. h–j, Three views of 12 Å resolution map of STING oligomers. Human STING CTD dimers bound to cGAMP were docked into the map. k, A list of human STING mutants in the transmembrane domain. l, Gel-filtration chromatography analyses of wild-type and mutants of full-length STING in the presence of 1 μM cGAMP using a Superose 6 column. m, SDS–PAGE analyses of fractions of wild-type STING and STING mutants purified by gel filtration chromatography using a superose 6 column. n, IFNβ luciferase reporter assays showing that the mutations in the N-terminal transmembrane domain affect STING-mediated signalling. Indicated amounts of pcDNA3.1-hSTING plasmids were transfected into HEK293T 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. NS, not significant. o, Western blot showing that mutations in the transmembrane domain of STING affect the phosphorylation of STING, TBK1 and IRF-3. HEK293T cells were transfected with indicated amounts of pcDNA3.1-hSTING plasmids and stimulated with cGAMP. p, Confocal microscopy images of HEK293T cells transfected with wild-type STING and STING mutants with or without cGAMP stimulation. Scale bars, 20 μm. Data in a–e, l and m are representative of at least two independent experiments. Data in f, g, o and p are representative of at least three independent experiments.
