The phosphorylation of STX17 on S202 by TBK1 releases STX17 from STING and distributes STX17 to distinct structures. (A) STING interaction with STX17 was inhibited TBK1 phosphorylation on STX17 S202. HEK293T cells were transfected with empty vector or STING-HA, combined with Flag-STX17-WT or Flag-STX17-S202A (SA) or Flag-STX17-S202D (SD) as indicated. 24 h after transfection, cells were lysed and immunoprecipitated with anti-HA agarose resin. (B) Flag-STX17-S202D co-localized with phagophore structure to a larger extend. U2OS cells were transfect with Flag-STX17 or Flag-STX17-S202A or Flag-STX17-S202D, combined with EGFP-DFCP1. After treated with Torin 1 for 3 h, cells were fixed and stained by indicated antibody and imaged by confocal microscopy. Representative images were shown. Scale bar, 10 µm. (C) Statistics result of B, representing co-localization between Flag-STX17-S202A or Flag-STX17-S202D with EGFP-DFCP1 in U2OS cells co-expressing plasmids as indicated and treated with Torin 1 for 3 h. At least 30 cells were analyzed. Data were mean ± SD. **, P ≤ 0.01. Unpaired two-tailed t test for comparison of two groups. (D) Flag-STX17-S202A better co-localized with complete autophagosome. U2OS cells were transfected with Flag-STX17 or Flag-STX17-S202A or Flag-STX17-S202D. After being treated with Torin 1 for 3 h, cells were fixed and stained by indicated antibody and imaged by confocal microscopy. Representative images were shown. Scale bars for both large and inset images, 10 µm. (E) Statistics result of D, representing co-localization between Flag-STX17-WT or Flag-STX17-S202A or Flag-STX17-S202D with overlapped LC3-Lamp2 area in U2OS cells expressing plasmids as indicated and treated with Torin 1 for 3 h. At least 30 cells were analyzed. Data were mean ± SD. ****, P ≤ 0.0001. Unpaired two-tailed T test for comparison of two groups. Source data are available for this figure: SourceData F8.
Figure 8.

The phosphorylation of STX17 on S202 by TBK1 releases STX17 from STING and distributes STX17 to distinct structures . (A) STING interaction with STX17 was inhibited TBK1 phosphorylation on STX17 S202. HEK293T cells were transfected with empty vector or STING-HA, combined with Flag-STX17-WT or Flag-STX17-S202A (SA) or Flag-STX17-S202D (SD) as indicated. 24 h after transfection, cells were lysed and immunoprecipitated with anti-HA agarose resin. (B) Flag-STX17-S202D co-localized with phagophore structure to a larger extend. U2OS cells were transfect with Flag-STX17 or Flag-STX17-S202A or Flag-STX17-S202D, combined with EGFP-DFCP1. After treated with Torin 1 for 3 h, cells were fixed and stained by indicated antibody and imaged by confocal microscopy. Representative images were shown. Scale bar, 10 µm. (C) Statistics result of B, representing co-localization between Flag-STX17-S202A or Flag-STX17-S202D with EGFP-DFCP1 in U2OS cells co-expressing plasmids as indicated and treated with Torin 1 for 3 h. At least 30 cells were analyzed. Data were mean ± SD. **, P ≤ 0.01. Unpaired two-tailed t test for comparison of two groups. (D) Flag-STX17-S202A better co-localized with complete autophagosome. U2OS cells were transfected with Flag-STX17 or Flag-STX17-S202A or Flag-STX17-S202D. After being treated with Torin 1 for 3 h, cells were fixed and stained by indicated antibody and imaged by confocal microscopy. Representative images were shown. Scale bars for both large and inset images, 10 µm. (E) Statistics result of D, representing co-localization between Flag-STX17-WT or Flag-STX17-S202A or Flag-STX17-S202D with overlapped LC3-Lamp2 area in U2OS cells expressing plasmids as indicated and treated with Torin 1 for 3 h. At least 30 cells were analyzed. Data were mean ± SD. ****, P ≤ 0.0001. Unpaired two-tailed T test for comparison of two groups. Source data are available for this figure: SourceData F8.

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