Figure 5: IFI16 interacts with cGAS but does not affect cGAMP production.

(a) IFI16 +/+ or IFI16 −/− HaCaT cells were stimulated with 5 μg ml⁻¹ HT DNA for the times indicated, and IFI16 was immunoprecipitated from cell lysates. Lysates and immunoprecipitates (IP) were analysed by SDS–PAGE and western blotting. (b) HEK293T cells were transfected with constructs for the expression of cGAS-FLAG and HA-IFI16, either wild-type (wt) or DNA-binding mutant (m4), as indicated. 24 h post transfection, cells were subjected to lysis and immunoprecipitation using FLAG antibody. Immunoprecipitates were washed, and treated with benzonase where indicated. Lysates and immunoprecipitates (IP) were analysed by SDS–PAGE and western blotting. (c) Multiple reaction monitoring transitions for cGAMP and cyclic di-AMP, used for the quantification of endogenous cGAMP and internal standard cyclic di-AMP. m/z, mass/charge ratio of fragment ions. (d) Standard curve for synthetic cGAMP spiked into cell lysates before sample preparation and liquid chromatography and mass spectrometry (LC-MS) analysis. (e) IFI16 +/+ and IFI16 −/− HaCaT cells were treated with 1 μg ml⁻¹ 70mer oligonucleotide or HT DNA for 8 h, followed by lysis in methanol, spike-in of c-di-AMP and sample preparation. cGAMP levels were determined by LC-MS, and normalized to c-di-AMP levels to account for losses in sample preparation and injection. Data are representative of at least four experiments; values are shown as mean of triplicate samples, with error bars representing s.d. (f) Total and extracted ion chromatogram of cGAMP and cyclic di-AMP in representative samples from (e), showing IFI16 +/+ and IFI16 −/− cells treated with HT DNA for 8 h. AA, integral peak area; RT, retention time.