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. 2024 Mar 11;15:2207. doi: 10.1038/s41467-024-46600-5

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© The Author(s) 2024

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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Fig. 4 — a Histogram of distances in light and heavy proteins in normalized fraction abundance profiles in normal and thapsigargin-treated (Thps.) AC16 cells. X-axis: the spatial distribution distance of two proteins is measured as the average euclidean distance of all TMT channel relative abundance in the ultracentrifugation fraction profiles across 3 replicates; y-axis: count. Blue: distance for 1,614 quantified light-heavy protein pairs (e.g., unlabeled vs. SILAC-labeled EGFR). Grey: distribution of each corresponding light protein with another random light protein. P value: Mann–Whitney test (two-sided). b Proportion of heavy-light protein pairs with confidently assigned localization to the same location (purple) in normal (left; 93%) and thapsigargin (right; 89%) cells. c Ranked changes in heavy-light pair distance in thapsigargin treatment. The positions of EGFR and ITGAV are highlighted. Inset: Z score distribution of all changes. d Spatial map of the light and heavy EGFR in normal and thapsigargin-treated cells. Each data point is a light or heavy protein species. Color: assigned compartment. Numbers: euclidean distance in fraction profiles over 3 replicates. e Corresponding fraction profiles; x-axis: ultracentrifugation fraction; y-axis: fractional abundance. Post-labeling synthesized EGFR is differentially distributed in thapsigargin and shows ER retention (blue), whereas the preexisting EGFR pool remains to show a likely cell surface localization (pink) after thapsigargin. f, g As above, for ITGAV. h Confocal imaging of EGFR immunofluorescence supports a partial relocalization of EGFR from the cell surface toward internal membranes following thapsigargin. Numbers: ratio of EGFR at the plasma membrane vs. whole cells. Blue: DAPI; green: EGFR; scale bar: 90 µm. i Cell areas; Mann-Whitney two-sided P: 0.72. Center line: median; box limits: interquartile range; whiskers: 1.5x interquartile range. j Total EGFR intensity per cell; Mann-Whitney two-sided P: 2.2e-05. Center line: median; box limits: interquartile range; whiskers: 1.5x interquartile range. k Edge/total intensity ratios in normal and thapsigargin-treated AC16 cells (n = 71 normal cells; n = 93 thapsigargin cells; Mann–Whitney two-sided P: 1.7e–4). Center line: median; box limits: interquartile range; whiskers: 1.5x interquartile range.