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. 2019 Feb 4;9:1161. doi: 10.1038/s41598-018-37894-9

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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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Identification of the PU.1 binding site. (a) Probes used in EMSAs. (b) The FLO-labeled Site 1&2 was incubated with the nuclear extracts prepared from JAWSII cells in the presence of either anti-PU.1 (PU) or non-specific (NS) Abs. (c) The FLO-labeled indicated probes were incubated with recombinant PU.1 protein in the presence of either anti-PU.1 (PU) or non-specific (NS) Abs. (d) The FLO-labeled indicated probes were incubated with recombinant PU.1 protein in the presence of 2-fold (×2) or 10-fold (×10) amounts of non-labeled identical WT or mutated competitor. After electrophoresis in 5% acrylamide gels, fluorescence was detected. (e) 293 T cells were transfected with either empty (mock) or PU.1-expression (PU.1) plasmids together with either WT or mutant reporter plasmids described in Fig. 1. At 48 h after transfection, luciferase and β-galactosidase activities were measured. Luciferase activities were normalized to β-galactosidase activities. Data are expressed as the ratio of the luciferase activity of the respective promoter-less plasmid-transfected cells. Results are shown as means + S.D.s (n = 3). Similar results were obtained in three independent experiments. *p < 0.05. Full-length gels with lower contrasts for (b–d), are included in Supplemental Information.