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. 2019 Mar 12;9:4246. doi: 10.1038/s41598-019-38982-0

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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 and preparation of Rv3463 protein. (A) Ammonium sulfate precipitate of Mycobacterium tuberculosis (Mtb) culture filtrate was fractionated with hydrophobic interaction chromatography (HIC) using Phenyl Sepharose. The primary fractions were sequentially fractionated by hydroxylapatite chromatography (HAT) and ion-exchange chromatography (IEC). (B) Fractions of interest were further separated by mini-whole gel eluter. (C) Recombinant Rv3463 purified from E. coli extracts was subjected to SDS-PAGE and western blot (WB) analysis using a mouse anti-His antibody. All proteins were analyzed by SDS-PAGE with Coomassie blue staining. (D) The cytotoxic effects of Rv3463 were analyzed by flow cytometry. BMDMs were stimulated with Rv3463, LPS or staurosporine (STS) for 24 h, and then stained with Annexin V and PI. The percentage of cells that are positive in each quadrant is indicated. The results are representative of three experiments.