Figure 6

pD345L-mediated inhibition of NF-κB signalling is independent of its exonuclease activity. A A schematic representation of the D345L catalytic activity mutant (D345L-CM), N-terminal mutant (D345L-N), and N-terminal deletion mutant (D345L-C). B 293 T cells transfected with IFNβ-Luc, pCMV-RL, pcDNA3.1-HA-cGAS and pcDNA3-2xFlag-STING, along with p3XFLAG-CMV-D345L, p3XFLAG-CMV-D345L-CM or empty vector for 24 h, were collected to measure the luciferase activity. C The experiment was performed as in Panel B except using pcDNA4-HA-TBK1. D 293 T cells transfected with NF-κB-Luc and pCMV-RL along with p3XFLAG-CMV-D345L, p3XFLAG-CMV-D345L-CM or empty vector for 24 h followed by treatment with TNFα (40 ng/mL) for 6 h were collected to measure the luciferase activity. E WSL cells transfected with p3XFLAG-CMV-D345L or p3XFLAG-CMV-D345L-CM or empty vector for 30 h followed by stimulation with 2′3′-cGAMP (5 μg/mL) for 4 h and harvested for RNA extraction. Semiquantitative RT–PCR assays were carried out to detect IFNβ, D345L and Actin mRNA expression levels. F The experiment was performed as for D, except that p3XFLAG-CMV-D345L-C was transfected. G The experiment was performed as for Panel E, except that p3XFLAG-CMV-D345L-C was transfected. 293 T cells were co-transfected with Flag-D345L, Flag-D345L-C and HA-IKKα (H), HA-IKKβ (I) or empty vector for 36 h, and the whole-cell lysates were immunoprecipitated with Flag antibody. The immunocomplexes were analysed by immunoblotting with the indicated antibodies.