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. 2021 Nov 26;9(5):nwab212. doi: 10.1093/nsr/nwab212

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© The Author(s) 2021. Published by Oxford University Press on behalf of China Science Publishing & Media Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 4. — BCAA increases USP1 protein via the GCN2-eIF2α pathway. (A) GCN2 inhibitor (GCN2iB, 5 μM) rescues the USP1 protein level with BCAA deprivation. ‘Ratio’, upper panel: USP1/β-actin; lower panel: Pi-eIF2α/eIF2α. (B) Histidinol (2 mM) decreases the USP1 protein level in a time-dependent manner. ‘Ratio’, upper panel: USP1/β-actin; lower panel: Pi-eIF2α/eIF2α. (C) Knockdown of eIF2α rescues the USP1 protein level under the BCAA deprivation condition. ‘Ratio’, upper panel: USP1/β-actin; lower panel: eIF2α/β-actin. (D) Overexpression of eIF2α wild-type but not enzyme-dead S51A mutant dramatically decreases the USP1 protein level. ‘Ratio’, upper panel: USP1/β-actin; lower panel: Flag/β-actin. (E) Tunicamycin (5 μg/mL) attenuates the USP1 protein level in a time-dependent manner. ‘Ratio’, upper panel: USP1/β-actin; lower panel: Pi-eIF2α/eIF2α. (F) ATF4 knockdown fails to rescue the USP1 protein level under the BCAA deprivation condition. ‘Ratio’, upper panel: USP1/β-actin; lower panel: ATF4/β-actin. (G) A higher BCAA diet decreases phosphorylation of eIF2α and has no effect on mTOR in KC mice. Mean ± SEM of n = 3 biologically independent experiments, two-tailed t-test. (H) Working model. BCAA deprivation activates the GCN2-eIF2α pathway and blocks USP1 protein synthesis. Data in (A–G) are representative of three biologically independent experiments. n.s. donates for no significance, ^*P < 0.05.