STING sequesters STX17 at ER in a stress dependent manner. (A) STING-HA interacts with endogenous STX17, but not SNAP29 and VAMP8. HEK293T cells were transfected with empty vector or STING-HA. 24 h after transfection, cells were lysed and immunoprecipitated with anti-HA antibody. (B) dSTING-HA interacts with dSYX17. HEK293T cells were transfected with empty vector, dSTING-HA, dSTX17-Flag with or without dSTING-HA. 24 h after transfection, cells were lysed and immunoprecipitated with anti-HA antibody. Immunoblotting was performed with indicated antibodies. (C) Direct binding of GST-STX17 to STING-His (139aa–379aa) and SNAP29 in vitro. Glutathione sepharose beads bound with GST or GST-STX17 were incubated with purified SNAP29 or STING-His (139aa–379aa) for 16 h and then eluted for immunoblotting. (D) Direct binding of STING-His (139aa–379aa) to Flag-STX17, but not Flag-SNAP29 or Flag-VAMP8 in vitro. Nickel NTA resin bound with STING-His (139aa–379aa) was incubated with Flag-STX17, or Flag-SNAP29, or Flag-VAMP8 for 16 h, and then eluted for immunoblotting. (E) STING-HA interacts with STX17-Flag, but not with Ykt6-Flag. HEK293T cells were transfected with empty vector, STX17-Flag, Ykt6-Flag with or without STING-HA. 24 h after transfection, cells were lysed and immunoprecipitated with anti-Flag antibody. Immunoblotting was performed with indicated antibodies. (F) Overexpression of STING-HA disrupts the interaction of STX17-Flag with SNAP29 and VAMP8. HEK293T cells were transfected with empty vector, STX17-Flag with or without STING-HA. 24 h after transfection, cells were lysed and immunoprecipitated with anti-Flag antibody. Immunoblotting was performed with indicated antibodies. (G) STING depletion increases STX17–SNAP29–VAMP8 SNARE complex formation. WT and STING-KO MEFs stable expressing STX17-Flag were lysed and immunoprecipitated with anti-Flag antibody and immunoblotting was performed with indicated antibodies. (H) STING blocks the translocation of STX17 to autophagosomes. WT MEFs, STING-KO MEFs, and STING reconstituted STING-KO MEFs with stable expression of STX17-Flag were treated with Torin 1 for 3 h and stained with antibodies against Flag, HA, and LC3. Scale bar for both large and inset images, 5 µm. (I) Quantification of the results in H. Percentage of LC3 dots with STX17. Data are mean ± SD for 100 cells. ***, P ≤ 0.001. Unpaired two-tailed t test for comparison of two groups. (J) The statistics result of Fig. S3 B indicates that STX17 co-localized with Calreticulin marked ER in STING WT or STING KO MEF cells expressing Flag-STX17 after treatment with Torin 1 for 3 h or no treatment, then cells were conducted immunofluorescence and imaging. Over 30 cells were analyzed. Data are represented as mean ± SD. *, P ≤ 0.05; ####, P ≤ 0.001. Unpaired two-tailed t test for comparison of two groups. (K) Interaction between STING-HA and STX17-Flag is decreased upon EBSS starvation, serum starvation, and Torin 1 treatment. HEK293T cells were transfected with empty vector, STING-HA with or without STX17-Flag. 24 h after transfection, cells were starved with EBSS, FBS deprived medium or treated with Torin 1 followed by immunoprecipitation with anti-Flag antibody. Source data are available for this figure: SourceData F7.
Figure 7.

STING sequesters STX17 at ER in a stress dependent manner. (A) STING-HA interacts with endogenous STX17, but not SNAP29 and VAMP8. HEK293T cells were transfected with empty vector or STING-HA. 24 h after transfection, cells were lysed and immunoprecipitated with anti-HA antibody. (B) dSTING-HA interacts with dSYX17. HEK293T cells were transfected with empty vector, dSTING-HA, dSTX17-Flag with or without dSTING-HA. 24 h after transfection, cells were lysed and immunoprecipitated with anti-HA antibody. Immunoblotting was performed with indicated antibodies. (C) Direct binding of GST-STX17 to STING-His (139aa–379aa) and SNAP29 in vitro. Glutathione sepharose beads bound with GST or GST-STX17 were incubated with purified SNAP29 or STING-His (139aa–379aa) for 16 h and then eluted for immunoblotting. (D) Direct binding of STING-His (139aa–379aa) to Flag-STX17, but not Flag-SNAP29 or Flag-VAMP8 in vitro. Nickel NTA resin bound with STING-His (139aa–379aa) was incubated with Flag-STX17, or Flag-SNAP29, or Flag-VAMP8 for 16 h, and then eluted for immunoblotting. (E) STING-HA interacts with STX17-Flag, but not with Ykt6-Flag. HEK293T cells were transfected with empty vector, STX17-Flag, Ykt6-Flag with or without STING-HA. 24 h after transfection, cells were lysed and immunoprecipitated with anti-Flag antibody. Immunoblotting was performed with indicated antibodies. (F) Overexpression of STING-HA disrupts the interaction of STX17-Flag with SNAP29 and VAMP8. HEK293T cells were transfected with empty vector, STX17-Flag with or without STING-HA. 24 h after transfection, cells were lysed and immunoprecipitated with anti-Flag antibody. Immunoblotting was performed with indicated antibodies. (G) STING depletion increases STX17–SNAP29–VAMP8 SNARE complex formation. WT and STING-KO MEFs stable expressing STX17-Flag were lysed and immunoprecipitated with anti-Flag antibody and immunoblotting was performed with indicated antibodies. (H) STING blocks the translocation of STX17 to autophagosomes. WT MEFs, STING-KO MEFs, and STING reconstituted STING-KO MEFs with stable expression of STX17-Flag were treated with Torin 1 for 3 h and stained with antibodies against Flag, HA, and LC3. Scale bar for both large and inset images, 5 µm. (I) Quantification of the results in H. Percentage of LC3 dots with STX17. Data are mean ± SD for 100 cells. ***, P ≤ 0.001. Unpaired two-tailed t test for comparison of two groups. (J) The statistics result of Fig. S3 B indicates that STX17 co-localized with Calreticulin marked ER in STING WT or STING KO MEF cells expressing Flag-STX17 after treatment with Torin 1 for 3 h or no treatment, then cells were conducted immunofluorescence and imaging. Over 30 cells were analyzed. Data are represented as mean ± SD. *, P ≤ 0.05; ####, P ≤ 0.001. Unpaired two-tailed t test for comparison of two groups. (K) Interaction between STING-HA and STX17-Flag is decreased upon EBSS starvation, serum starvation, and Torin 1 treatment. HEK293T cells were transfected with empty vector, STING-HA with or without STX17-Flag. 24 h after transfection, cells were starved with EBSS, FBS deprived medium or treated with Torin 1 followed by immunoprecipitation with anti-Flag antibody. Source data are available for this figure: SourceData F7.

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