Extended Data Fig. 3: C-178 and C-176 covalently bind to Cys91.

a, BMDMs were treated with C-178 (0.25 μM) for 1 h, washed or left untreated and after 1 h were stimulated with cGAMP for 3 h. Levels of Ifnb1 mRNA were assessed by RT–qPCR. Data are mean ± s.e.m. (n = 3 biological replicates). b–d, HEK293T cells expressing indicated Flag–mmSTING constructs were exposed to C-178 or C-176 (1 μM), lysed and Flag–mmSTING was immunoprecipitated. The eluted protein was analysed by intact mass spectrometry (LC-MS). b, Deconvoluted electrospray ionization mass spectrum (left), and expected and measured mass shifts after covalent binding of C-176 and C-178 (right) are shown. c, Deconvoluted electrospray ionization mass spectrum for Flag–mmSTING(C91S). d, Fragmentation map of Flag–mmSTING–C-176 analysed by top-down analysis, using LC-HCD-MS/MS (an additional 8 residues are due to N-terminal Flag). Data that were obtained with three different NCE values are combined to create a fragmentation map with assigned b- and y-fragments. Achieved sequence coverage is 15% with 20 p.p.m. mass accuracy for fragment assignment. One representative of n = 2 independent experiments is shown (b, c). For electrospray ionization spectra see Supplementary Information.