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. 2021 Jan 20;12:476. doi: 10.1038/s41467-020-20780-2

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© The Author(s) 2021

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. 4 — a, b Co-IP experiments (a) and quantitative results (b) showed that phosphorylation of ENDOG at T128/S288 is necessary for its interaction with 14-3-3γ (overexpression of wild-type and mutant ENDOG in the ENDOG knockout L02 cell; WT: wild-type; DD: T128D/S288D; AA: T128A/S288A; n = 3 independent experiments; data are presented as mean values ± SD, *p < 0.05; **p < 0.01). c, d Representative immunofluorescence images of GFP-LC3 puncta (c) and respective quantitative results (d) (ENDOG knockout cells transiently transfected with the wild-type and mutant ENDOG for 48 h and treated with or without 100 nM BafA1 for 6 h; scale bar = 10 μm; n = 100 independent cells examined over three independent experiments; data are presented as mean values ± SD; **p < 0.01, ***p < 0.001). e, f Western blots (e) and quantitative results (f) of mTOR pathway and autophagy-related proteins. ENDOG-knockout cells were transiently transfected with the wild-type and mutant ENDOG for 48 h (n = 4 independent samples; data are presented as mean values ± SD; **p < 0.01, ***p < 0.001). g, h. Co-IP experiments (g) and quantitative results (h) showed that GSK-3β phosphorylates ENDOG and enhances the interaction between ENDOG and 14-3-3γ (transiently transfected of Myc-ENDOG, wild-type GSK-3β or kinase dead mutant GSK-3β-K85A in ENDOG knockout L02 cells for 48 h; n = 3–4 independent experiments; data are presented as mean values ± SD; **p < 0.01, ***p < 0.001). i, j Co-IP experiments (i) and quantitative results (j) showed that GSK-3β phosphorylates ENDOG at T128 and S288 (transiently transfected of Myc-ENDOG, Myc-ENDOG-AA or GSK-3β in ENDOG-knockout L02 cells for 48 h; n = 4 independent experiments; data are presented as mean values ± SD; **p < 0.01). k In vitro phosphorylation of ENDOG by GSK-3β by using the recombination protein (ATP: 5 μM; n = 4 independent experiments; data are presented as mean values ± SD, **p < 0.01). Source data are provided as a Source data file.