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. 2023 Apr 12;616(7958):764–773. doi: 10.1038/s41586-023-05927-7

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© The Author(s) 2023

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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Extended Data Fig. 14 — a. BioID2 that is targeted with Glt1-BioID2 biotinylates proteins at astrocyte sites of glutamate uptake. b. Label-free based quantification comparison of significant proteins (Log₂FC > 1 and FDR < 0.05 versus GFP controls) detected in the cytosolic Astro BioID2 and Astro Glt1-BioID2 reveal Glt1 enriched proteins. Top half of the volcano plot shows 527 unique Glt1-BioID2 proteins when compared to cytosol. The top four most abundant proteins for Glt1-BioID2 are shown. Lower half of volcano plot shows comparison of 230 proteins that were common in both cytosolic BioID2 and Glt1-BioID2. The five highest enriched proteins for Glt1-BioID2 are shown. Magenta label shows protein that was validated with immunohistochemistry. c. Heat map shows the rank-rank hypergeometric overlap (RRHO) of the RNA and protein rank for the 532 Glt1-BioID2 proteins. Each pixel represents the significance of overlap between the two datasets in –log₁₀(P-value). Red pixels represent highly significant overlap. Color scale denotes the range of P-values at the negative log₁₀ scale (bin size = 100). d. IHC analysis of Faim2 protein in tdTomato and Glt1-GFP labeled astrocytes shows co-localization within the astrocyte territory. Scale bar represents 20 μm. e. Co-localization analysis using Pearson’s r co-efficient shows high co-localization between Glt1-GFP and Faim2. The mean and SEM are shown (n = 8 tdTomato+ cells from 4 mice; Two-tailed paired t-test). f. Scale-free STRING analysis protein-protein association map of the top 100 unique and enriched biotinylated proteins identified with Astro Glt1-BioID2 . Node size represents the enrichment of each protein vs the GFP control (log2(BioID2/GFP)). Edges represent putative interactions from the STRING database. Bar graphs show the functional enrichment analysis of all 532 proteins using “Biological process”, “Cellular component”, and “Molecular function” terms from Enrichr. The image of the astrocyte subcompartments in panel a was created using BioRender (https://www.biorender.com/).