Figure 2. Palmitoylation at Cys88/91 of STING is required for the type I interferon response.

(a) emsCOS-1 cells were starved for 1 h, followed by the incubation with [³H] palmitate for 1 h. Cells were then stimulated with DMXAA for the indicated times. Cell lysates were prepared and EGFP–STING was immunoprecipitated with anti-GFP antibody. Cell lysates and the immunoprecipitates were analysed by western blot and autoradiography. (b) Primary MEFs were examined as in a. For immunoprecipitation, anti-STING antibody was used. (c) The effect of 50 μM 2-BP on the palmitoylation of STING in emsCOS-1 cells. (d) Quantitative real-time PCR (qRT-PCR) of the expression of IFNβ in emsCOS-1 cells that were pretreated with vehicle or 50 μM 2-BP for 1 h and then stimulated with DMXAA for 12 h. (e,f) qRT-PCR of the expression of IFNβ in primary MEFs (e) or BMDMs (f) that were pretreated with vehicle or 50 μM 2-BP for 1 h and then stimulated with ISD for 3 h. (g) COS-1 cells that stably express EGFP-mouse STING with indicated Cys mutations were metabolically labelled, stimulated with DMXAA, and analysed by western blot and autoradiography. (h) The band intensities in g were quantified and [³H palmitate]/[GFP] were calculated. The data are normalized to the value of WT-STING with DMXAA treatment and represent mean±s.e.m. of two independent experiments. (i) COS-1 cells that stably express STING (C88/91S) were stimulated with DMXAA for 1 h, fixed, permeabilized and stained for TGN46 (red). Nuclei were stained with DAPI (blue). Scale bars, 10 μm. (j) COS-1 cells that stably express STING (WT or C88/91S) were stimulated with DMXAA for 12 h. qRT-PCR of the expression of IFNβ was then performed. (k) Primary Sting^−/− MEFs were reconstituted with human STING variants using retroviruses. The cells were stimulated with ISD for 6 h, and qRT-PCR of the expression of IFNβ was performed. Data in d,e,f,j and k are mean±s.e.m. from three independent experiments. *P<0.01, ***P<0.001, NS, not significant (one-way analysis of variance).