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. 2020 Sep 21;31(2):157–177. doi: 10.1038/s41422-020-00409-1

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© The Author(s) 2020

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. 3 — a Left, immunofluorescence of EGFR-HA (red) and Flag-RAB31^Q65L (magenta) with CD63-GFP (green) in Flag-RAB31^Q65L stable HeLa cells stably expressing shNC (negative control), shFLOT1, shFLOT2 or shFLOT1 and shFLOT2 and transiently expressing EGFR-HA and CD63-GFP under serum starvation (SS). Right up panel, the ratio of entry of EGFR-HA into CD63-GFP-positive LE/MVE in shNC (n = 9 fields), shFLOT1 and shFLOT2 (n = 9 fields), shFLOT1 (n = 12 fields), shFLOT2 (n = 12 fields). Right low panel, the ratio of entry of Flag-RAB31^Q65L into CD63-GFP-positive LE/MVE in shNC (n = 9 fields), shFLOT1 and shFLOT2 (n = 9 fields), shFLOT1 (n = 12 fields), shFLOT2 (n = 12 fields). b Western blotting analyses of the concentrated conditional media from the indicated stable HeLa cells used in a. c Up panels, immunofluorescence of EGFR-HA (red) and Flag-RAB31 (magenta) with FLOT1-GFP (green) in the indicated stable HeLa cells transiently expressing EGFR-HA and FLOT1-GFP under SS. Low panel left, the ratio of co-localization of EGFR-HA with FLOT1-GFP-positive vesicle in Vector (n = 7 fields), RAB31^WT (n = 8 fields) and RAB31^Q65L (n = 9 fields). Low panel right, the ratio of co-localization of Flag-RAB31 with FLOT1-GFP-positive vesicle in RAB31^WT (n = 8 fields) and RAB31^Q65L (n = 9 fields). d Up panels, immunofluorescence of FLOT1-HA (red) and Flag-RAB31 (magenta) with CD63-GFP (green) in the indicated stable HeLa cells transiently expressing FLOT1-HA and CD63-GFP under SS. Low panel left, the ratio of co-localization of FLOT1-HA with CD63-GFP-positive LE/MVE in Vector (n = 7 fields), RAB31^WT (n = 7 fields) and RAB31^Q65L (n = 8 fields). Low panel right, the ratio of entry of FLOT1-HA into CD63-GFP-positive LE/MVE in Vector (n = 7 fields), RAB31^WT (n = 7 fields) and RAB31^Q65L (n = 8 fields). e Left, immunofluorescence of EGFR-HA (red) and Flag-RAB31^Q65L (magenta) with CD63-GFP (green) in Flag-RAB31^Q65L stable HeLa cells transiently expressing EGFR-HA and CD63-GFP and treated with DMSO, 5 μM GW4869, 5 μM simvastatin or 10 μM lovastatin under SS. Right up panel, the ratio of entry of EGFR-HA into CD63-GFP-positive LE/MVE in DMSO (n = 8 fields), GW4869 (n = 11 fields), simvastatin (n = 13 fields) and lovastatin (n = 12 fields). Right low panel, the ratio of entry of Flag-RAB31^Q65L into CD63-GFP-positive LE/MVE in DMSO (n = 8 fields), GW4869 (n = 11 fields), simvastatin (n = 13 fields) and lovastatin (n = 12 fields). f Western blotting analyses of the concentrated conditional media from the indicated stable HeLa cells used in e. All data are means ± SD. Unpaired t-test was used to analyze the difference between the two groups. ****P < 0.0001, ***P < 0.001, NS, no statistical significance. Scale bars, 10 μm.