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. 2019 Sep 20;10:4320. doi: 10.1038/s41467-019-12309-z

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© The Author(s) 2019

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. 8 — OTULIN binding to SNX27 limits recycling of GLUT1 and SLC1A4 to the cell surface. a GLUT1 surface staining in mock, OTULIN WT and OTULIN ΔETSL transfected U2OS cells was performed using GLUT1.RBD.GFP reagent on living cells. GLUT1 surface expression was analyzed in untransfected (ΔCD2-low) and in transfected (ΔCD2-high) cells by FACS (left). Relative GLUT1 surface expression was determined as depicted in the histogram and changes in median fluorescence intensity (MFI) were normalized to mock. Graphs represent the mean ± SD of three independent experiments. Two-tailed p-values: ns not significant, ***p ≤ 0.001 by unpaired t-test. b U2OS cells lentivirally transduced with GFP-SLC1A4 (green) together with RFP, RFP-OTULIN, or RFP-OTULIN ΔETSL (gray) were stained for endogenous LAMP1 (red) and analyzed for SLC1A4/LAMP1 co-localization by confocal microscopy. Scale bars: 10 µm. c Co-localization was quantified by determination of Pearson’s correlation analyzing at least 14 random pictures and imaging of more than 100 cells for each condition. Graphs depict the mean ± SD. Two-tailed p-values: ns not significant, ***p ≤ 0.001 by unpaired t-test. d Knock-down in U2OS cells after siRNA transfection was verified by WB. e GLUT1 surface staining in U2OS cells after siRNA transfection as indicates was performed using GLUT1.RBD.GFP reagent on living cells. GLUT1 surface expression was analyzed by FACS. f Changes in median fluorescence intensity (MFI) were normalized to siRNA control to calculate relative GLUT1 surface expression. Graphs represent the mean ± SD of three independent experiments. Two-tailed p-values: ***p ≤ 0.001 by unpaired t-test. g Knock-down in HeLa cells after siRNA transfection was verified by WB. h HeLa cells transfected with siRNA as indicated and stained for endogenous GLUT1 (red) and the lysosomal marker LAMP1 (green). Co-localization of GLUT1 and LAMP1 was analyzed by confocal microscopy. Scale bars: 10 μm. i Co-localization was quantified by determination of Pearson’s correlation analyzing at least eight random pictures and imaging of more than 100 cells for each condition. Graphs depict the mean ± SD. Two-tailed p-values: ns not significant, ***p ≤ 0.001 by unpaired t-test. j Schematic model for the dual function of OTULIN. Through LUBAC binding and catalytic activity OTULIN controls Met1-ubiquitin chain homeostasis. By binding to SNX27 OTULIN counteracts cargo recruitment and retromer assembly to antagonize endosome-to-plasma membrane trafficking of internalized cargos. Source data are provided as a Source Data file