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. 2017 Dec 19;7:17753. doi: 10.1038/s41598-017-18101-7

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© The Author(s) 2017

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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EMSA for in vitro binding of Snail to the proposed Snail binding region on MMP9 promoter. Nuclear extracts obtained from HepG2 were treated with 50 nM TPA for the times indicated, followed by EMSA using MMP9-proSN (lanes 2-5). Unlabed wild-type or mutant competitors in 200X amount were included in the EMSA for HepG2 treated with TPA for 2 h (lanes 6 and 7). Lane 1 is the sample of probe only. For antibody blocking analysis, each of the indicated antibody was preincubated with the nuclear extract from HepG2 treated with TPA for 2 hr followed by EMSA reaction (lanes 8–10). Schematic map of the EMSA probes (MMP9-porSN), and the competitor (MMP9-porSN) located around Snail binding motif upstream of the EGR-1/SP1 overlapping region on MMP9 promoter is demonstrated in supplemental Fig. 2C.