Identifying the binding site of STING for STX17 and effects of STING mutants on autophagy inhibition. (A) Scheme of WT STING, cyclic dinucleotides (CDNs) binding-deficient mutant STING R232A, and autoimmunity disease mutants STING N154S or STING V155M. (B) Overexpression of STX17-myc has no effect on STING self-interaction. HEK293T cells were transfected with empty vector, STING-HA, STING-Flag, STX17-Myc as indicated. 24 h after transfection, cells were lysed and immunoprecipitated with anti-Flag antibody. Immunoblotting was performed with indicated antibodies. (C) STX17-HA interacts with STING-Flag through the region around 134–160aa of STING. HEK293T cells were transfected with empty vector, STX17-HA and Flag tagged STING truncations. 24 h after transfection, cells were lysed and immunoprecipitated with anti-Flag resin. Immunoblotting was performed with indicated antibodies. White arrows indicate designated bands of truncations of STING. (D) The cartoon of 2D STING structure. Three residues important for STX17 interaction are marked in red. (E) Representative images of LC3 puncta in cells expressing STING WT or the indicated mutants. WT MEFs, STING-KO MEFs, STING-KO MEFs stably expressing STING-HA WT, STING-HA R232A, STING-HA N154S, STING-HA V155M, or STING-HA C148A were treated with Torin 1 for 3 h and then stained with antibodies against HA and LC3. Scale bar, 10 µm. Source data are available for this figure: SourceData FS4.