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. 2018 May 15;9:1920. doi: 10.1038/s41467-018-04244-2

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© The Author(s) 2018

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. 5 — ESRRG directly antagonizes Wnt signaling. a, b AGS or MKN28 (b) cells were infected with Flag or Flag-ESRRG-lentiviral vector. The cells (a) including fractionated samples (b) were also used for western blotting with the indicated antibodies. c, d The infected AGS (c) and MKN28 (d) cells were treated with CHX for the indicated times and the samples were used for western blotting with the indicated antibodies. e ChIP assays were performed on AGS or MKN28 cells after transfection with ESRRG using a TCF4/LEF1 antibody. Recruitment of ESRRG to the CCDN1 promoter via TCF4/LEF1 was analyzed using primers specific to the CCND1 promoter. f ChIP assay was done with ESRRG antibody. IgG was used as an internal control. g Immunoprecipitation was done in MKN28 cells with the indicated antibodies and detected with Flag antibody. h, i, l GC cells or organoids (l) were transfected with GFP-ESRRG and mCherry-TCF4 or LEF1 and used for cellular imaging under a confocal microscope. j Changes over time in the average fluorescence intensities (count per second; c.p.s in kHz) of GFP-ESRRG (green) and mCherry-TCF4 or LEF1 (red), and the corresponding correlation functions, are shown. Changes over time in the average fluorescence intensity and the corresponding correlation functions obtained in cells co-expressing monomer GFP and mCherry are also shown. k Summary of protein interaction amplitudes. The interaction amplitude represents the mean value of the relative cross-correlation amplitude. Data represent the mean ± s.d. from three independent replicates. Student’s t-test was used to examine statistical significance (*p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001)