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. 2018 Dec 3;8:17536. doi: 10.1038/s41598-018-35616-9

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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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Chk1 T378/382 auto-phosphorylation is not augmented by DNA damage or DNA synthesis inhibition. (a) Chk1 REV DT40 cells stably expressing Chk1-WT, or Chk1-S345A DT40 cells stably expressing a mutant where alanine replaces S345 (see text for additional explanation), were treated for 16 hours with ETOP and HU in the presence or absence of MG132. Cell extracts were prepared, 30 μg resolved by SDS-PAGE, and analysed by western blotting using the indicated antibodies. Samples were resolved on three separate western blots (see Supplementary Information for original images). (b) CETSA analysis of the thermal stability of the Chk1-WT, Chk1 L393P, and Chk1-DD proteins in cell extracts. Protein expression was induced by treatment with DOX for 16 hours and native, soluble whole cell extracts prepared¹⁴. Portions of each extract were heated at 55 °C for 5 minutes or retained on ice. Insoluble proteins were then removed by centrifugation and 30 μg resolved by SDS-PAGE in triplicate and analysed by western blotting using antibody recognising total Chk1. Shown is the mean and standard deviation of the percentage of soluble Chk1 remaining after treatment at 55 °C.