Extended Data Fig. 6: NF-κB activation via TRAF6-IRF3-STING pathway is conserved in terrestrial animals.
From: STING signals to NF-κB from late endolysosomal compartments using IRF3 as an adaptor
a, FLAG was immunoprecipitated from HEK293T cells co-transfected with Myc-TRAF6 and different species of STING, and stimulated with vehicle or cGAMP for 6 h. The precipitates were immunoblotted with anti-Myc. b, Quantification of Myc-TRAF6 by western blotting. The band intensity of Myc-TRAF6 was plotted after normalization to the Flag-STING signal of the same lane. c, The activation of STING-NF-κB mediated by TRAF6-IRF3 is conserved in terrestrial animals. The mechanism of regulation involves IRF3 docking at STING, which acts as an adaptor to recruit TRAF6. This mechanism is highly conserved from amphibians to mammals. The affinity level between STING and IRF3 determines the preference of species STING to activate IFN or NF-κB. In lower species, STING has a higher affinity for IRF3, leading to the induction of a higher level of NF-κB response. In contrast, higher species STING conversely exhibit a lower propensity. d, Phylogeny analysis of type I interferon genes in terrestrial animals. e, HaCaT cells deficient in STING were reconstituted with human/mouse STING-WT or mutants as indicated. The cells were stimulated with cGAMP delivered by Digitonin and then incubated at 37 °C or 21 °C for 4 h. The levels of the indicated proteins were determined by immunoblotting. f, PBMCs from carp and mouse were stimulated with vehicle or cGAMP at 37 °C or 21 °C. Total RNA was isolated and analyzed by RT-qPCR. Results are presented as mean ± SD (n = 3). Results presented in a, b, e, f are representative from 3 independent experiments with similar results.
