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. 2022 Jun 1;13:3041. doi: 10.1038/s41467-022-30803-9

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© The Author(s) 2022

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. 7 — a Expression profiles of Gid4 and Gid12 tagged at their endogenous loci, under different growth conditions, monitored by western blotting against hemagglutinin tag (HA). YPD: glycolytic conditions; YPE: overnight non-fermentable carbon source starvation; Carbon Recovery: glucose replenished with YPD. Pgk1 served as loading control (n = 3 biologically independent experiments). b Degradation of 3×HA chromosomally tagged Gid4 in wildtype (WT) and Gid12-overexpressing (GPD promoter) cells were monitored using anti-HA immunoblotting. Gid4 was stable under carbon recovery conditions in a strain continuously overexpressing Gid12 (n = 3 biologically independent experiments). c Quantitative mass spectrometry analysis of the relative levels of Gid2, Gid4 and Gid12 in anti-HA immunoprecipitates of lysates expressing Gid4 with an N-terminal 3×HA tag at the endogenous loci. Yeast cells were grown under glycolytic (YPD), non-fermentable carbon source starvation (YPE), or carbon recovery conditions. Data are mean ± s.d. of n = 3 biologically independent experiments. iBAQ ratios are formed using the respective Gid2 iBAQ value as denominator. d Monitoring degradation of 3×FLAG chromosomally tagged gluconeogenic enzymes in wildtype (WT), Gid12-deficient (gid12∆), and Gid12-overexpressing (ADH1 promoter) cells using anti-FLAG immunoblotting. The effects of GID12 deletion to gluconeogenic enzymes are subtle, however, all four gluconeogenic enzymes are stabilized under carbon recovery conditions in a strain consecutively overexpressing Gid12 (n = 3 biologically independent experiments).