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. 2017 Dec 11;8(12):3201. doi: 10.1038/s41419-017-0070-z

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© The Author(s) 2017

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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Fig. 1 — a Yeast two-hybrid assay. L40 cells were transformed with LexA DNA binding domain (DBD)-fused SIRT1 (in pBTM116 vector) and Gal4 activating domain (AD)-fused CACUL1. The interaction was measured by β-galactosidase (β-gal) assays. The data were processed in accordance with statistical analysis methods as means±standard deviation (SD) of triplicate experiments. b Immunoprecipitation (IP) analysis. HEK293 cells were co-transfected with Myc-SIRT1 and FLAG-CACUL1 or FLAG vector. IP using an anti-FLAG antibody was followed by western blotting (WB) with an anti-Myc antibody. c Glutathione S-transferase (GST) pull-down assay. GST or GST-CACUL1 was incubated with in vitro translated FLAG-SIRT1. The interaction was monitored by WB using an anti-FLAG antibody. d, e Mapping of domains responsible for the interaction. HEK293 cells were transfected with green fluorescent protein (GFP)-CACUL1 and FLAG-tagged SIRT1 deletions, or Myc-SIRT1 and GFP-fused CACUL1 fragments. SIRT1 mapping was determined by IP using an anti-FLAG antibody and WB using an anti-GFP antibody (d). CACUL1 mapping was done by IP using an anti-Myc antibody and WB using an anti-GFP antibody (e)