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. 2023 Aug 10;25(9):1303–1318. doi: 10.1038/s41556-023-01198-6

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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 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. 3 — a,b, Fasnall treatment does not influence mTORC2 activity in HEK293FT cells. Cells were treated with 25 μM Fasnall or DMSO (–) as the control for 30 min (n = 4 independent experiments). a, Immunoblotting for AKT phosphorylation at S473 was used to assess mTORC2 activity. b, Levels of mTORC2 activity (p-AKT^S473/AKT). c,d, Effect of cerulenin treatment (50 μM, 4 h) on cells as in a,b (n = 3 independent experiments). d, Levels of mTORC2 activity (p-AKT^S473/AKT). e–i, Neither expression of the acc1^S1157A allele nor treatment of yeast cells with cerulenin (20 μM, 2 h) downregulates TORC2 or Snf1 (yeast AMPK) activity. e, Lysates from control (−) or cerulenin-treated wild-type (WT) and acc1^S1157A mutant cells were immunoblotted with the indicated antibodies (n = 3 independent experiments). Phosphorylation of Ypk1 was used as the TORC2 readout. Phosphorylation of Snf1 was used as the AMPK activation readout. Total Snf1 was detected with an antibody to a histidine (His) stretch in Snf1. Mal-K blots showing total protein malonylation, which is indicative of intracellular Mal-CoA levels. f–i, Levels of TORC2 activity (p-Ypk1^T662/Ypk1; f), TORC1 activity (p-Sch9^T737/Sch9; g), Snf1 activation (p-Snf1^T210/His; h) and lysine malonylation (Mal-K/Adh1; i). j, Inhibition of FASN downregulates mTORC1 activity independently of lipid availability. Immunoblots with lysates from control (−) or Fasnall-treated (25 μM, 30 min) HEK293FT cells supplemented with BSA-conjugated FAs, as indicated, or BSA as a control. Phosphorylation of S6K and 4E-BP1 was used to assay mTORC1 activity (n = 2 independent experiments). b,d,f,g,h,i, Data are the mean ± s.e.m. *P < 0.05; **P < 0.005; ***P < 0.0005; and NS, not significant. Ctrl, control; and Ceru, cerulenin. Source numerical data and unprocessed blots are provided.

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