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. 2023 Sep 30;13:182. doi: 10.1186/s13578-023-01130-3

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© The Author(s) 2023

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/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 6 — The extent of HIF-1α acetylation and HIF-1α interaction with VHL is increased during chronic hypoxia via SIRT1 inactivation. (A ~ C) HeLa cells co-transfected with Flag-tagged HIF-1α and either HA-tagged VHL (HA-VHL) were exposed to hypoxic conditions for 9 and 24 h (A and B) or 6 h (C). The effect of pyruvate (A), Myc-tagged SIRT1 (B), and either SIRT1- or control-shRNA (-) transfection (C) on HIF-1α interaction to VHL (A ~ C), acetylation, (B and C) was monitored in immunoprecipitated HIF-1α preparations, followed by probing with anti-acetyl-K and/or anti-HA antibodies, respectively. (D ~ E) The contribution of an association of VHL with HIF-1α in terms of HIF-1α stability was determined in the presence of SIRT1- (+) or control-siRNA (-), or pyruvate in RCC4 and RCC4/VHL cells. (E) The extent of acetylation of immunoprecipitated HIF-1α was assessed in RCC4 and RCC4/VHL cells treated with NAM. (F-G) Acetylation and ubiquitination of immunoprecipitated HIF-1α were assessed in RCC4/VHL cells treated with pyruvate alone (F), or with concomitant addition of NADH 6 h before harvest (G). The concentration of MG132, pyruvate and NADH was 10 mM, 1 mM, and 1 mM, respectively