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. 2017 Jun 28;8:15865. doi: 10.1038/ncomms15865

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Copyright © 2017, The Author(s)

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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(a,b) Immunoblot analysis for RIP2 from various cellular fractions of HCT116 cells stimulated with MDP for 30 min. The cells were fractionated to separate (a) cytoplasmic (CF), membrane (MF) or (b) plasma membrane (PM), endoplasmic reticulum (ER) fractions, respectively. Equal amount of each fraction (30 μg) was analysed by western blot. Relative densitometric data (using ImageJ) of RIP2 levels in various fractions is shown in the bottom panels (n=3). (c) Confocal microscopy of HCT116 cells expressing RIP2-HA (red), YFP-ZNRF4 (green) and pmTurquoise2-ER (ER marker-blue). EV, vector control. Images were acquired at × 63 optical magnification. Scale bars, 10 μm. Western blots and confocal images are representative of one of three independent experiments that were conducted. *P<0.05. Significance was assessed using Student’s t-test. si-NT, non-targeting negative control siRNA. Protein disulfide isomerase (PDI)=marker for ER; cadherin (pan)=marker for PM.