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. 2022 Oct 20;13:6212. doi: 10.1038/s41467-022-33951-0

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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. 5 — a HeLa cells were stained with antibodies for FLOT1, FLOT2, and LAMP2 followed by microscopy imaging (n = 3). Lower panels display a zoom-in of the regions indicated in the full picture. Mander’s coefficients show the average degree of overlap of one protein population relative to another. Data are presented as mean values + SD (n = 3, cells examined over one experiment). Profile plots indicate the degree of colocalization for individual vesicles. b Analysis of FLOT1/FLOT2 in early endosomes (n = 1). Following pulsed SPIONs treatment, endosomes were enriched at the indicated chase times. Pre-CTSD serves as a marker for early endosomes. c Western blot analysis of early endosome-enriched fractions with marker proteins for different subcellular compartments (n = 1): Cytosol (ACTG2 and GAPDH), cytoskeleton (TUBA), endoplasmic reticulum (CANX), and mitochondria (SDHA). d Experimental workflow for early endosome enrichment and XL-LC-MS/MS analysis. Created with BioRender.com. e Overview of the cross-linking dataset obtained from early endosome-enriched fractions. f Overlap of unique FLOT1/FLOT2 cross-links for XL-LC-MS/MS analyses of early endosome- and lysosome-enriched fractions. g Western blot analysis of BN-PAGE-separated FLAG-IP eluates with/without cross-linking by DSSO (n = 5). IN/EL refers to individual FLAG-IP fractions. h Western blot analysis of SDS-PAGE separated FLAG-IP ELs with/without DSSO cross-linking for WT and FLOT1/FLOT2 residue 200–300 deletion mutants (Δ100) (n = 3). Scale bar = 5 µM; WCL whole cell lysate, IN input, WA wash, EL eluate, MCOE Mander’s coefficient, Dst. distance, LS lysosomes, EE early endosomes, XL cross-linking, SPIONs superparamagnetic iron oxide nanoparticles, M marker, WT wild type. Source data are provided as a Source Data file.