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. 2018 Aug 9;8:11929. doi: 10.1038/s41598-018-30166-6

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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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Electrophoretic mobility shift assay (EMSA) by recombinant mTERF6. (A) Scheme of the probes used in the EMSA assay. Black boxes, white boxes and straight lines represent the exons, introns and interval regions, respectively. The locations and sequences of probes A and B were 5′-end labeled with biotin. Probe A is a double-stranded DNA fragment produced by PCR amplification and purification. Probe B is a single strand DNA. (B) Expression and purification of MBP-mTERF6.1 by SDS-PAGE analysis. Lanes 1 and 2 are the total proteins from the pre-induction cultures and post-induction cultures (induced by 0.4 mM IPTG at 18 °C for 6 h), respectively; lane 3 is the soluble extract induced (by 0.4 mM IPTG at 18 °C) overnight; lane 4 is the purified recombinant MBP-mTERF6.1 protein. The arrow indicates the expected band (This is a cropped gel. Full-length gels are included in Original pictures of Supplementary Fig. 6B). (C) The binding of MBP-mTERF6.1 to probe A. “fp” represents free probe. (D) The binding of MBP-mTERF6.1 to probe B. (E) Comparison of the exact binding sites of rpoA (3′-end) and trnI.2 and the 3′-end DNA sequences of other genes (rbcL, ycf5 and petD). Red “*” indicates that the analyzed DNA sequences of ycf5 are inside its downstream adjacent gene.