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. 2017 Jul 6;8:15979. doi: 10.1038/ncomms15979

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Copyright © 2017, The Author(s)

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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(a–c) Interaction between DNA and MLDS was detected by single-molecule pull-down assay. Biotin-labelled DNA was incubated with Cy5-labelled MLDS, and the mixture was immobilized onto the streptavidin-coated coverslips and Cy5-labelled MLDS was observed. Sketch (a), validation (b) and quantification (c) of single-molecule pull-down assay were shown. The Cy5-labled MLDS non-specificly bound to the PEG-passivated surface is shown in left. Scale bar, 3.5 μm. Data represent mean±s.d., n=3. **P<0.01, two-tailed t-test. (d) EMSA analysis of the interaction between DNA and MLDS. A dose dependent binding of GST-MLDS was obtained. Although these gel shifts were diffused when the protein/DNA ratio was from 20 to 40, these bindings could still be gradually increased since free monomer DNA was decreased with increased protein, and the number of GST-MLDS molecules bound to the 2.5 kb DNA could be saturated at 220 per DNA molecule (red). (e) The amino acid sequence of the MLDS C-terminus (MLDS^C) and the MLDS C-terminal mutant (MLDS^C′). All lysine in the region was replaced by glutamic acids. (f) EMSA analysis of MLDS^C and MLDS^C′ with DNA. The 2.5 kb DNA molecule bound a maximum of 360 molecules of MLDS^C (red). (g) The morphology and model of adiposomes. (h,i) Sketch (h) and validation (i) of adiposome-binding assay using adiposomes, DNA, and MLDS or MLDS N-terminus (MLDS^N). DNA, proteins and adiposomes were incubated and then the reaction was centrifuged to separate the adiposomes from the solution. DNA was detected by PCR and EB stained agarose gel (bottom). Protein was detected by silver staining (top). Lanes 4 and 9: MLDS^N, DNA and adiposomes; lanes 6 and 11: MLDS, DNA and adiposomes. Lanes 2–6 represented adiposome samples; lanes 7–11 represented solution samples. MLDS^N and MLDS represented GST- MLDS^N and GST-MLDS, respectively. (j) Similar to i, validation of adiposome-binding assay using adiposomes, DNA, and MLDS or JLP. Lanes 2 and 10: JLP, DNA and adiposomes; lanes 5 and 13: MLDS, DNA and adiposomes. JLP represented GST-JLP.