Fig. 5: cGAS phosphorylation by DNA-PK inhibits the activity of cGAS.

a cGAS^−/− L929 cells were infected with control (Vec) lentivirus or that containing cGAS WT or the phosphorylation-mimic mutants. WCLs were analyzed by immunoblotting. b Reconstituted cGAS stable cells as described in a were selected with puromycin (5 μg/ml) for 3 days. The expression of the indicated inflammatory genes was analyzed by real-time PCR. c Reconstituted cGAS stable cells were transfected with HT-DNA (1 μg/ml). Cells were harvested at 6 h post transfection, and the expression of the indicated inflammatory genes was analyzed by real-time PCR. d 293T cells were transfected with WT or the mutant cGAS plasmids. WCLs were precipitated with anti-FLAG agarose. Precipitated proteins and WCLs were analyzed by immunoblotting with the indicated antibodies. e, f Purified cGAS WT or mutants were analyzed by Coomassie blue staining (e). In vitro cGAMP activity assay was performed without or with HT-DNA (1 μg/ml) stimulation and the activity was presented as the enzymatic activity percentage against WT cGAS (f). All experiments were done at least twice, and one representative is shown. n = 3 biologically independent samples for b, c, f. Data are presented as mean values ± SD. Source data are provided as a Source data file.