In vitro ubiquitination assay

Purified His-STING (152–379 aa, 500 nM) was mixed with GST-MUL1 (300 nM), UBE1 (100 nM), UBE2D1 (1 μM), ubiquitin (50 μM) (UBPBio) in a reaction buffer containing 20 mM Tris–HCl, pH 7.6, 50mM NaCl, 5 mM MgCl₂, 2mM ATP, 1 mM β-ME and 5% glycerol. The reaction was carried out at 37°C for 2 hr and then subjected to SDS-PAGE followed by immunoblot with indicated antibodies.