Figure 3. Analysis of CnpB 3’3’-c-di-AMP binding and hydrolysis.

(a) Thin layer chromatography (TLC) analysis of enzymatically synthesized [³²P] 3’3’-c-di-AMP and [³²P] ATP standard. Data are representative of several independent experiments. (b) DRaCALA analysis of 3’3’-c-di-AMP binding to RECON and CnpB in presence and absence of 0.1 mM MnCl₂. data are presented as mean ± standard deviation of n=3 replicates. (c-d) TLC analysis of 3’3’-c-di-AMP hydrolysis by CnpB. (c) ~1 nM [³²P] 3’3’-c-di-AMP was incubated with or without 25 μM CnpB in the presence and absence of 0.1 mM MnCl₂ for 2 hours at 37°C. Samples were then treated with or without 0.1 U of Alkaline Phosphatase for 1 hour. Samples were then spotted onto TLC plates and separated. Data are representative of n=3 experiments. (d) 1 μM unlabeled 3’3’-c-di-AMP spiked with ~1 nM [³²P] 3’3’-c-di-AMP was treated with or without 1 μM CnpB overnight at 37°C. Samples were then spotted onto TLC plates and separated. N=3 replicates are shown.