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. 2017 Jul 4;8:15948. doi: 10.1038/ncomms15948

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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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Eight key residues that caused dysfunction of Lnt in the in vivo assay were combined in the C23A/C62A variant of Lnt (that is, WT*) and were analysed by mal-PEG alkylation. Lnt (C23A/C62A/C387A), that is, C387A*, lacks any cysteine residue and was used as a negative control. Whole-cell lysates were treated without (a) or with (b) 1 M neutral hydroxylamine (which reduces all thioester bonds) and subsequently treated with mal-PEG. Samples were analysed by SDS-10% polyacrylamide gel electrophoresis followed by immunoblotting with antibodies against polyHis-tag. Alkylated (Lnt-mal-PEG) and non-alkylated forms of Lnt are indicated. Note that, variants E267A, K335A, E343A, C387A and E389A have been reported to be inactive using the same assay¹⁸.