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. 2021 Jun 15;28(12):3251–3269. doi: 10.1038/s41418-021-00813-z

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© The Author(s) 2021

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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — A LoVo and SW620 cells were lysed and subjected to immunoprecipitation with anti-ATG9B antibody or rabbit IgG using silver staining. The red boxes represents the detected bands. B Endogenous ATG9B and MYH9 were immunoprecipitated in SW620 cells. C Exogenous ATG9B^His and MYH9^Flag were immunoprecipitated in 293T cells. D ATG9B colocalized with MYH9 in SW480/ATG9B cells. The magnified parts are displayed in the lower right panel and the arrows points to the colocalized sites (scale bar 10 μm, r (Person’s correlation coefficient) = 0.72). E Cytoplasmic segments of ATG9B, including aa1–207 (Cd1), aa298–438 (Cd2), aa548–551 (Cd3) and aa646–924 (Cd4). F Diagrammatic representation of ATG9B and its truncated forms. Based on sequence and structure analyses, region I (cytoplasmic domain), region II (transmembrane domain) and region III (luminal domain) are indicated. 293T cells were transfected with the indicated constructs subjected to immunoprecipitation with anti-Flag (against MYH9). Immunoblot analysis was performed with anti-Flag or anti-His (against ATG9B). The red boxes represents the pull-down bands. G Direct binding of MYH9^Flag to ATG9B-Cd2^His using His pulldown assay. The red boxes represents the pull-down bands. H Constructs with deleted mutations Δ298-332, Δ333-367, Δ368-411 and Δ412-438 of ATG9B-Cd2^His were used for immunoprecipitation with MYH9^Flag. I Diagrammatic representation of MYH9 and its truncated forms. 293T cells were transfected with the indicated constructs subjected to immunoprecipitation with anti-His (against ATG9B). The red boxes represents the pull-down bands. J Transwell invasion assay detected SW480 cells transfected with vector, ATG9B^His together with MYH9^Flag or ATG9B^Δ368-411 together with MYH9^Flag (scale bar 50 μm, n = 5). Data information: Graphs report mean ± SEM. Significance was assessed using two-tailed Student’s t test. ***P < 0.001, **P < 0.01, *P < 0.05.