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. 2020 Apr 6;10:5971. doi: 10.1038/s41598-020-62912-0

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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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Electrophoretic mobility shift (EMSA) and reporter assay. (a) To assess the ability of the recombinant FL-CXXC5 or its CXXC domain (CXXC-D), 50 μM DNA with the central unmethylated (CG) or methylated (mCG) CpG dinucleotides was mixed with FL-CXXC5 at 1:0.5, 1:1, 1:2, 1:4 and 1:8 molar ratios or with CCCX-D at 1:4 molar ratios. Samples were run onto 5% native TBE gel, stained and visualized with UV spectrometry. “M” represents the molecular marker. “Free” denotes unbound DNA; “Shifted” indicates DNA bound protein. A representative experiment performed with two independent times is shown. (b) To assess the intrinsic transcription regulatory function of FL-CXXC5, pGal4-RE Luciferase Reporter vector (pGal4RE-Luc, 125 ng) containing tandem Gal4 response elements (Gal4-RE) juxtaposed to a simple TATA box promoter that drives the expression of the firefly Luciferase enzyme cDNA together with an expression vector (75 ng) bearing Gal4_DBD, VP16, FL-CXXC5, WT-MeCP2 or ERα-EF domain cDNA or Gal4_DBD-VP16, Gal4_DBD-CXXC5, Gal4_DBD-MeCP2 or Gal4_DBD-ERαEF cDNA transfected into MCF7 cells. MCF7 cells grown in 10% CD-FBS for 48 h were transfected with expression vector bearing ERαEF cDNA or Gal4_DBD-ERαEF and were treated without (%0.01 ethanol) or with 10⁻⁸ M E2 for 24 h. Transfection efficiency was monitored with the co-expression of a reporter plasmid bearing the Renilla Luciferase enzyme cDNA (0.5 ng). Results indicating relative firefly/Renilla luciferase activity determined using a dual luciferase assay kit are the mean ± S.E. of three independent experiments performed in duplicate. The asterisk denotes significant differences compared to responses observed with Gal4-RE, which is set to 1. (c) Cells were also transfected with pGal4RE-Luc together with the expression vector bearing none (empty vector, EV), Gal4_DBD-VP16 and/or Gal4_DBD-CXXC5 cDNA. In transfections, we used a total of 300 ng expression vector (1 denotes 75 ng), for which appropriate amounts of the parent expression vector (EV) were supplemented to equalize the total plasmid DNA amount. Results presented as relative firefly/Renilla luciferase levels indicate percent change and are the mean ± S.E. of three independent experiments performed in duplicate. The asterisk denotes significant differences from responses observed with Gal4_DBD-VP16, which was set to 100%.