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. 2017 May 15;8:15560. doi: 10.1038/ncomms15560

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Copyright © 2017, The Author(s)

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

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(a) FLAG-tagged p65 was transiently co-expressed with V5-tagged full-length Sirt1, truncated Sirt1 (ΔCTD Sirt1), Clover or Clover-CTD fusion proteins into 293 HEK cells as indicated. Forty-eight hours post transfection, cells were incubated with either 25 mM Glu or 25 mM 2-DG as indicated for 2 h. Cell extracts were immunoprecipited with anti-V5 antibody and then immunoblotted with either anti-FLAG for detection of the amount of p65 or anti-V5 for immunoprecipitants. ΔCTD and CTD indicate the Sirt1 fragments that span from 1 to 510 amino acids and from 510 to 737 amino acids of mouse Sirt1, respectively. (b) The experiment performed in a was repeated after transfections of V5-tagged Clover and Clover-CTD expression vectors into 293 HEK cells. Cells were treated for 2h with either 25 mM Glu or 25 mM 2-DG as indicated, 48 h post transfection. The binding amount of p53 with Clover or Clover-CTD was visualized by immunoprecipitating with anti-V5 antibody and immunoblotting with p53 antibody. (c) FLAG-tagged p65 was transiently co-expressed with V5-tagged Clover, Clover-CTD or Clover-ΔESA and co-immunoprecipitation was performed after treatment with either Glu or 2-DG as in a. ΔESA indicates CTD missing the ESA region (amino acids from 631 to 655). (d) V5-tagged Sirt2 or Sirt2-CTD was transiently co-expressed with FLAG-tagged p65 and co-immunoprecipitation was performed as above after treatment with either Glu or 2-DG. Sirt2-CTD indicates the CTD of Sirt1 is linked to the end of C-terminal end of full-length of Sirt2 as a fusion protein. (e,f) To visualize the effect of Sirt1 CTD has on deacetylase activity of Sirt2, Sirt2 or Sirt2-CTD was co-expressed with either (e) FLAG-tagged p65 or (f) p53 and treated with either Glu or 2-DG. Acetylated p65 was visualized by immunoprecipitation of p65 using followed by immunoblotting with anti-acetyl p65 (K310) antibody. Acetylated p53 level was visualized by immunoblotting with anti-acetyl-p53 (K382) antibody.

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