Fig 6. Neddylation promotes the activation of cGAS.

(A) HEK293T cells were transfected with indicated plasmids. 24h after transfection, cells were stimulated with HT-DNA for 4h before cells were harvested for cGAMP detection. (B) Rnf111 deficiency or control BMDM were transfected with ISD for 4h, cell lysates were heat denatured, and cGAMP was measured by ELISA. (C) Ube2m deficiency BMDMs were infected with HSV-1 (MOI = 0.5) for 3h, after adding human cDNA as an external reference, cell lysates were immunoprecipitated with anti-cGAS antibody, then cGAS-bound DNA was extracted and quantified by qRT-PCR by normalized to human GAPDH. (D) Rnf111 deficiency BMDMs were infected with HSV-1 (MOI = 0.5) for 3h, after adding human cDNA as an external reference, cell lysates were immunoprecipitated with anti-cGAS antibody, then cGAS-bound DNA was extracted and quantified by qRT-PCR by normalized to human GAPDH. (E) Rnf111 deficiency BMDMs were transfected with an empty plasmid for 3h, after adding human cDNA as an external reference, cell lysates were immunoprecipitated with anti-cGAS antibody, then cGAS-bound DNA was extracted and quantified by qRT-PCR by normalized to human GAPDH. (F) HEK293T cells were transfected with indicated plasmids for 45h and then infected with HSV-1 (MOI = 0.3) for 4h, the cell lysates were immunoprecipitated with an anti-Flag antibody and then immunoblotted with indicated antibodies. (G) HA-NEDD8 and Flag-cGAS were coexpressed in HEK293T cells, 48h later cells were collected and cell lysates were incubated with recombinant His-cGAS, the interaction was analyzed by immunoprecipitated with anti-Flag beads and immunoblotted with indicated antibodies. (H) Poly-NEDD8 chains were generated in vitro, followed by incubating with His-cGAS. After immunoprecipitated with anti-His antibody, and then immunoblotted with indicated antibodies. (I) Poly-NEDD8 chains were generated in vitro, followed by incubating with His-cGAS. After immunoprecipitated with anti-NEDD8 antibody, and then immunoblotted with indicated antibodies. Graphs are presented as means ± SEM, data are representative of three independent experiments, *P <0.05; **P <0.01 (One-way ANOVAs followed by Tukey’s post hoc test).