Extended Data Fig. 5: 2’3’-cGAMP initiates and promotes STING phase separation and gel-like transition.
From: The STING phase-separator suppresses innate immune signalling
a, The Coomassie staining (left) and western blot (right) of indicated purified proteins were shown. STING EE/GG, STING E336G/E337G. Antibodies against STING, TBK1 and IRF3 were used. b, Phase-separation diagram of STING and NaCl at indicated concentrations at 37°C in a phase separation buffer (50 mM Tris-HCl, 750 mM NaCl, 10% BSA, 5% PEG, 0.15% SLS, pH 7.5). Green dots: Phase separation; white dots: no phase separation; black dots: inaccessible conditions (purified STING protein can be homogenous at most to 3 μM in 2 M NaCl buffer at room temperature leading to some inaccessible ionic conditions). Images represented all fields in wells. c, Representative fluorescence confocal images showing fusion of STING droplets. STING phase separation was performed as in Fig. 3a (37°C). d, Representative images of STING phase separation reconstitution of different STING concentration at the physiological condition (50 mM Tris-HCl, 150 mM NaCl, 0.03% SLS, pH 7.5, 37°C). STING concentration was increased by centrifugation as in Methods. STING droplets in 0.1 μM and 1.5 μM were imaged under x100 objective while condensates in 40 μM was too large and needed to be imaged under x40 objective. e, Representative confocal images of STING droplets formed by in vitro reconstitution using purified WT or E336G/E337G mutant STING with the same protein concentration at 4°C as in Fig. 3a (4°C) for 12 h. f, Phase-separation diagram of human STING and NaCl after mixing with CDN (c-di-GMP or 2’3’-cGAMP) at indicated concentrations at 37°C in the phase separation buffer (50 mM Tris-HCl, 750 mM NaCl, 10% BSA, 5% PEG, 0.15% SLS, pH 7.5) for 10 min. Green dots: phase separation; white dots: no phase separation; red dots: gel-like transition. For all Extended Data Fig. 5, data were representatives of at least three independent experiments. Confocal images represented all fields in a well. Scale bar, as indicated. See uncropped gel images in source data.
