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. 2020 Dec 31;10:22436. doi: 10.1038/s41598-020-80288-z

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© The Author(s) 2020

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Generation of stable CRC cell clones expressing NAT2 slow acetylator variants. (A) Two clones encoding each of the indicated NAT2 slow acetylator variants were chosen for downstream analyses based on their expression of NAT2 RNA relative to parental RKO cells. Data from one representative experiment shown. (B) Sanger sequencing revealed the correct 341 T > C, 857 G > A, and 191G > A substitutions in the NAT2 coding region (black arrows) to generate the NAT2*5, *7 and *14 alleles. (C) Protein expression of NAT2 slow acetylator variants by immunoblotting. A Myc-tag antibody was used to detect recombinant NAT2 with β-actin as loading control. Full-length immunoblots are presented in Supplementary Fig. 4. (D) Quantification of immunoblots shown in (C). Protein expression levels were normalized against β-actin.