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. 2019 Dec 30;9:20301. doi: 10.1038/s41598-019-56748-6

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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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Involvement of RNF183 in the localization and degradation of DR5. (a) Effect of RNF183 overexpression on DR5 subcellular localization. HeLa cells transfected with EmGFP-tagged RNF183 or EmGFP alone were subjected to immunofluorescence staining with DR5 and LAMP1 antibodies (grey, EmGFP; green, DR5; red, LAMP1). (b) Effect of RNF183 knockdown on DR5 protein levels. RNF183-siRNA or non-target control (NC)-siRNA were transfected into HEK293 cells stably expressing V5-tagged RNF183. Cell lysates were subjected to western blotting with DR5 and V5 antibodies. The full-length blots are presented in Supplementary Fig. S10. (c) Quantitative graph of data from (b). Asterisks represent significant differences (n = 3; paired t-test, ***p < 0.001). (d) Cell surface expression level of DR5. HEK293 cells expressing V5-tagged RNF183 were treated with 100 μM CQ for 12 h. Then, cells were labeled with PE-conjugated anti-DR5 antibody. (e) DR5 protein levels in colonic epithelial cells from DW- and DSS-treated mice at 5 days. Cell lysates were subjected to western blotting with anti-DR5 and anti-β-actin antibodies. The full-length blots are presented in Supplementary Fig. S11. (f) Quantitative graph of data from (e). Asterisks represent significant differences (n = 6; paired t-test, **p < 0.01).