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. 2025 Feb 6;16:1407. doi: 10.1038/s41467-025-56720-1

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© The Author(s) 2025

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, 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 you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. 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-nc-nd/4.0/.

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Fig. 4 — a Workflow of BioID2 approach illustrating biotinylation of eSHANK3-interacting proteins, purification of the biotinylated proteins by NeutrAvidin agarose beads, mass spectrometry analysis, and validation through IP analysis. Created in BioRender. Kim, S. (2025) https://BioRender.com/q75n212. b ICC analysis using anti-HA antibody and Alexa Fluor® 488-Streptavidin shows colocalization of eSHANK3-BioID2-HA (or BioID2-HA) and biotin labeled proteins only in the biotin-treated bEnd.3 BECs. Arrowheads indicate the co-expression of SHANK3^Δe18-BioID2 and biotinylated proteins in the cell membrane. Scale bars, 50 μm. Blue: DAPI. c Streptavidin blot analysis, using purified biotinylated proteins, showed the protein bands of various sizes that were biotinylated by BioID2-HA or Shank3^Δe18-BioID2-HA. Four biological replicates were analyzed, all yielding consistent results. 5% of purified proteins were loaded for the blotting analysis. 95% of purified proteins were used for subsequent mass spectrometry analysis. d Mass spectrometry analysis identified 376 candidate eSHANK3 interacting proteins. Node size represents protein abundance [log₂ (fold change)] over eSHANK3^Δe18-only (no BioID2) and BioID2-only control groups. Solid gray edges delineate interactions between the eSHANK3 and the identified interactome. Dashed edges indicate known interactions. e GO analysis revealed membrane and junctional proteins are the primary eSHANK3 interactome in the bEnd.3 BECs. f IP analysis confirmed the interactions between endogenous eSHANK3 and some of the identified interactome in the bEnd.3 BECs. Three times independent experiments yielded similar results. IgG: control for non-specific antibody binding. Heavy chain IgG: loading control. The detailed information of the identified eSHANK3 interactome and GO analysis are provided in Supplementary Data 1. Source data are provided as Source Data File.