Figure 4. cGAMP binds to STING and activates IRF3 in a STING-dependent manner.

(A) Increasing concentrations of HT-DNA or cGAMP were delivered to indicated cells and the induction of IFNβ was measured by q-RT-PCR. Inset shows immunoblots of STING and β-tubulin in the cell lines. (B) Indicated cell lines were infected with HSV1Δ34.5 or permeabilized with digitionin and then incubated with cGAMP. Activation of endogenous IRF3 was analyzed by native gel electrophoresis (top). Aliquots of the cytosolic extracts were heated to denature proteins, and the supernatant was assayed for its ability to stimulate IRF3 in permeabilized Raw264.7 cells (bottom). (C) cGAMP, c-di-GMP, ISD, or poly[I:C] was delivered into L929 cells stably expressing a shRNA against GFP or STING for the indicated time, followed by analysis of IRF3 dimerization. (D) Recombinant STING protein was incubated with [³²P]-ATP or [³²P]-cGAMP in the presence or absence of the cold competitors as indicated. After UV crosslinking, the mixtures were resolved by SDS-PAGE followed by autoradiography.