Figure 3.

Optimization of ENPP1 cleavage of cGAMP to yield quantitation limit of 62.5 nm. A, cGAMP standard curve with high-zinc ENPP1 buffer quantitated by cGAMP-Luc assay. Data are mean ± S.E. (error bars) (n = 3). B, AMP standard curve quantitated by an AMP-Luc assay (n = 1; data representative of two independent experiments). R² is 0.9945 for 0 μm Zn²⁺, 0.9955 for 50 μm Zn²⁺, and 0.9648 for 100 μm Zn²⁺. C, 2′,3′-cGAMP hydrolysis reactions. Products of reactions were submitted for analysis by LC-MS/MS. Data are mean ± S.D. (error bars) (n = 2). D, AMP standard curve with low-zinc ENPP1 buffer quantitated by an AMP-Luc assay. Data are mean ± S.E. (n = 2). E, cGAMP standard curve with low-zinc ENPP1 buffer quantitated by cGAMP-Luc assay. Data are mean ± S.E. (n = 4). F, a serial dilution of ENPP1 inhibitors QS1 and 1084 was incubated with 10 nm ENPP1 and 5 μm cGAMP for 2 h at room temperature. The resulting AMP was quantitated via the AMP-Luc assay (n = 3 for 1084, n = 2 for QS1).