In vitro interaction between STING and biotinylated Ap4A was tested using Dynabeads M280, blocked as described above. Ten micromolar of biotinylated Ap4A (BioLog) or biotin was bound to the beads at 25°C for 30 min in 2× wash buffer. Thirty, 50, or 100 μg of recombinant proteins (mSTING, mHINT, and GST) was incubated with the beads for 1 hour at 4°C on the wheel in a final volume of 200 μl in 150 mM NaCl, 50 mM tris-HCl (pH 8.0), 10 mM MgCl2, BSA (0.1 μg/μl), 10 mM β-mercaptoethanol, and 0.5 mM PMSF. Competition was performed after protein binding using a 10-fold excess of the competitor (DMXAA, 2′3′-cGAMP, Ap4A, or JB419) for 1 hour at 4°C on wheel in a final volume of 200 μl in the buffer described above. After three washes in 150 mM NaCl, 50 mM tris-HCl (pH 8.0), 10 mM MgCl2, 0.1% Tween, BSA (0.1 μg/μl), 10 mM β-mercaptoethanol, and 0.5 mM PMSF, bound material was eluted in 30 μl of Laemmli buffer.

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