Fig. 4

O-GlcNAcylation of STING on T229 promotes its K63-linked Ubiquitination. A O-GlcNAcylation residues in STING were analyzed and predicted with the Yin-O-Yang website. B and C A series of mutant constructs of STING were generated with mutations in individual residue predicted by the Yin-O-Yang website. STING in the pull-down complexes and in the WCEs was detected with immunoblotting (B). (C) STING and its mutants were immunoprecipitated with anti-Flag antibody. O-GlcNAcylated Flag-STING and its mutants were detected with an anti-O-GlcNAc monoclonal antibody, RL2. STING and its mutants in WCEs were detected with immunoblotting. D Immunoblotting of K63 ubiquitination in immunoprecipitated complex pulled down with STING antibody from lysates of cells treated with or without DON (10 μM) or TMG (10 μM) for 12 h. E HEK-293 T cells were transfected with FLAG-tagged STING-WT or -T229A in the presence of His-tagged ubiquitin. Co-IP was performed with an anti-STING antibody from abcam. Immunoblotting was performed with antibodies against His-tag (His-Ub), K63-Ub, RL2, and STING. Data are representatives from 3 independent experiments