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. 2022 Oct 16;13:6112. doi: 10.1038/s41467-022-33881-x

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© The Author(s) 2022

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. 1 — a The indicated FLAG-tagged proteins were stably expressed in 293^L cells and non-detergent extracts subjected to immunoprecipitation with α-FLAG antibodies prior to proteomic analysis of trypsinized peptides using 10-plex TMT-based proteomics which included one replicate each of immunoprecipitates and whole-cell extracts in the 10-plex. The enrichment (Log₂FC) of endosomal proteins³² (gray circles) relative to control 293^L cells is shown, with FLAG-EEA1 displaying the greatest enrichment of proteins known to localize to endosomes. Left border, interior line, and right border in the box plot represent the 1st quartile, median, and 3rd quartile, respectively. b 293^EL cells (clone 33) were subjected with immunofluorescence using α-FLAG (green, to detect FLAG-EEA1) and α-RAB5 (magenta) antibodies, and nuclei were stained with Hoechst 33342 (blue). Image analysis of 11 cells indicates that the Mander’s coefficient is ~0.7 for both the overlap of FLAG puncta with RAB5 and RAB5 puncta with FLAG. c Scheme depicting affinity purification of PI3P-positive early endosomes using FLAG-EEA1 in conjunction with α-FLAG antibodies immobilized on magnetic beads. d Control 293 cells or 293^EL cells (clone 33) were lysed in non-denaturing buffer prior to either direct analysis of extracts by immunoblotting or were subjected to α-FLAG magnetic bead capture followed by immunoblotting with the indicated antibodies. Loaded amounts are equivalent to 0.06% and 6% of the input and IP eluate, respectively. e α-FLAG immunoprecipitates from either control 293 cells or 293^EL cells (clone 33) were released from the affinity resin by FLAG peptide and then analyzed by transmission EM. Arrowheads indicate vesicular structures present in cells expressing FLAG-EEA1 but not control cells. Scale bar, 150 nm.