FIGURE 4.

WT/SAVI heterocomplex does not activate the STING signalling. (A) EGFP-WT and FLAG-WT were stably expressed in Sting ^−/− MEFs. Cells were stimulated with or without DMXAA (25 μg ml⁻¹) for 2 h. Cells were then fixed, permeabilized, and immunostained with anti-p-STING antibody and anti-FLAG antibody. The boxed areas are magnified in the bottom panels. The fluorescence intensity profiles along the magenta lines are shown. The red arrows indicate the puncta positive with EGFP-WT, FLAG-WT, and p-STING. (B) EGFP-SAVI (V154M) and FLAG-WT were stably expressed in Sting ^−/− MEFs. Cells were incubated with BFA (0.3 μg ml⁻¹) for 3 h followed by 1 h incubation without BFA. Cells were then fixed, permeabilized, and immunostained with anti-p-STING antibody and anti-FLAG antibody. The boxed areas are magnified in the bottom panels. The fluorescence intensity profiles along the magenta lines are shown. The red arrows indicate the puncta positive with EGFP-SAVI (V154M) and p-STING. (C) HEK293T cells were transfected with the plasmids encoding EGFP-STING and FLAG-STING as indicated. Cell lysates were prepared, and FLAG-STING was immunoprecipitated with anti-FLAG beads. The cell lysates and the immunoprecipitated proteins were analysed by western blot. (D). HEK293T cells were transfected with the plasmids encoding Myc-STING and FLAG-STING as indicated. Cell lysates were analysed by western blot. (E) HEK293T cells were transfected with the plasmids as indicated, together with an ISRE (also known as PRDIII or IRF-E)-luciferase reporter. Luciferase activity was then measured. Data represent mean ± s.e.m. of three independent experiments. Scale bars, 10 µm.