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. 2018 Feb 14;9(2):246. doi: 10.1038/s41419-018-0281-y

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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. 7 — a Immunoprecipitation assay shows the functional interaction of Nanog and Hdac4. 293T cells were transfected with expression vectors for Nanog and FLAG-Hdac4. Cell lysates were subjected to immunoprecipitation with anti-FLAG antibody or normal IgG as a negative control, and the precipitates were subjected to immunoblot analysis with anti-Nanog or anti-FLAG antibodies, respectively. Five percent of the whole-cell lysate was loaded for the input control. b Immunostaining of undifferentiated P19 cells with specific antibodies shows the subcellular localization of Nanog and Hdac4. Hoechst dye was used for staining nuclei. c, d Nanog overexpression facilitates the recruitment of Hdac4 onto Gata4 and Nkx2.5 promoters. Undifferentiated P19 cells were transfected with plasmids encoding Nanog. After 48 h of transfection, cells were collected and ChIP assays were conducted with antibodies against Nanog (c) and Hdac4 (d). Immunoprecipitated DNA fragments were amplified by real-time PCR for the promoter regions of Gata4 and Nkx2.5 (S1 region). Each bar represents mean ± SD from three independent experiments (*p < 0.05; #p < 0.01 vs. the control)