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. 2024 Jan 20;15:631. doi: 10.1038/s41467-024-44978-w

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© The Author(s) 2024

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — a PI(3)P production was analyzed by the ELISA assay. The fold change was calculated based on the concentration of PI(3)P and normalized against control group. b The effect of serum starvation (S.S.) on STX18-Flag and ATG14-HA interaction was analyzed by co-immunoprecipitation. c GST pull-down analysis of GST-ATG14 with STX18-His. d The effect of STX18-HA expression on the interaction of Flag-LC3C with ATG14-HA was analyzed by immunoprecipitation. e The effect of STX18 on the interaction of LC3C with ATG14 was detected by GST pull-down. f The effect of STX18 knockdown on the interaction of Flag-LC3C with ATG14-HA was analyzed by immunoprecipitation. g, h Co-localization of GFP-ATG14/LC3/LDs in Stx18 knockout HeLa cells expressing indicated plasmids were treated with 200 μM OA for 12 h. 100 μM CQ was added for 6 h before cells were fixed. The number of colocalization of ATG14/LC3/LDs per cell was counted from 50 cells of three independent experiments. i HEK293T cells were transfected with STX18-Flag. Protein interactions were detected by immunoprecipitation with anti-Flag beads and immunoblotting analysis. j The effect of STX18-Flag expression on the interaction of ATG14-HA with Myc-Vps34 and Myc-Beclin1 was analyzed by immunoprecipitation. k Vps34 complex (Vps15, Vps34, ATG14, Beclin1) was purified from 293 F cells and incubated with or without STX18-Flag and analyzed via Chromatography of C1 on Superose 6 10/300 GL columns. l HEK293T Stx18 wild-type and knockout cells were transfected with indicated plasmids. Protein interactions were detected by immunoprecipitation. m HeLa cells were transfected with ATG14 or/and STX18 siRNA for 48 h. Cell lysis were analyzed via western blot. n, o HeLa cells expressing GFP-FYVE_SARA were transfected with ATG14 or/and STX18 siRNA for 48 h, and treated with 200 μM OA for 12 h. Co-localization of LDs and GFP-FYVE_SARA. The number of colocalization of PI3P with LDs per cell in (n and Supplementary Fig. 4j) was counted from 50 cells of three independent experiments. Data information: Scar bar represents 10 μm for all fluorescence images. Error bars, mean ± SD of three independent experiments. Two-tailed Unpaired Student’s t test. Source data are provided as a Source Data file.