Extended Data Fig. 5: IRF3 Ser396 and the TRAF6 binding motif synergize to mediates STING-induced NF-κB activation.
From: STING signals to NF-κB from late endolysosomal compartments using IRF3 as an adaptor
a, HaCaT cells deficient in IRF3 were rescued with IRF3 WT, R211Q, R285Q, S386A, or S396A. and stimulated with vehicle or cGAMP. The levels of the indicated proteins were determined by immunoblotting. b, Murine embryonic fibroblasts (MEFs) were treated with control (scrambled gRNA)- or TRAF6-targeting gRNAs. The cells were treated with cGAMP for 6 h and total RNA was isolated for RT-qPCR analysis. Data show normalized levels of Tnfa and Il6 mRNA measured by RT-qPCR (n = 3). c, THP1 cells were treated with control (scrambled gRNA)- and TRAF6-targeting gRNAs and treated with cGAMP for 2 h. Lysates were probed for the indicated proteins measured by immunoblotting. d, SDS-PAGE for verification of protein purification and chromatogram of size exclusion chromatography of TRAF6-MATH domain or IRF3 peptide. e, Schematic of STING chimera CTT constructs with/without TRAF6 binding motif from human IRF3 or fish STING CTT. f, FLAG was immunoprecipitated from HEK293T cells with FLAG-STING WT/ + TRAF6 binding motif transfected with Myc-TRAF6. Precipitates were immunoblotted with anti-Myc. g, NF-κB/ISRE-driven reporter assay. HEK293T cells expressing STING-WT/STING + TRAF6 binding motif were co-transfected with 50 ng of the NF-κB/ISRE promoter luciferase reporter and 30 ng of the β-actin Renilla reporter (n = 3). h, WT and IRF3-deficient HEK293T cells were co-transfected with Flag-tagged TRAF6 and HA-tagged Ub-K63 only. After transfection for 24 h, the cells were stimulated with vehicle or cGAMP. Co-immunoprecipitation and immunoblot analysis were performed with the indicated antibodies. i, HEK293T cells with stable expression of STING and IRF3-WT or IRF3 mutants as indicated were transfected with Flag-tagged TRAF6. FLAG was immunoprecipitated from the cells, and the precipitates were immunoblotted with the indicated antibodies. Results are presented as mean ± SD (b and g). P values were calculated using a two-sided, two-way ANOVA with Tukey’s multiple comparisons test (b and g). All results presented in this figure are representative from 3 independent experiments with similar results.
