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. 2023 Mar 3;14:1214. doi: 10.1038/s41467-023-36881-7

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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. 4 — YY2 protein expression in Tsc2−/− 105K cells (a) and Tsc2 KO MEFs (b) treated with 20 nM rapamycin (Rapa) or vehicle for 24 hr (n = 3). YY2 protein expression in Tsc2−/− 105K cells (c) and Tsc2 KO MEFs (d) treated with 10 μM PF-4708671 (S6K1 inhibitor) or vehicle for 24 hr (n = 3). e Immunoblot analysis of HeLa cells transfected with eGFP-YY2, wild-type S6K (HA-WT-S6K), or kinase-dead S6K (HA-KD-S6K) for 48 hr (n = 3). f Immunoblot analysis of in vitro kinase assays of recombinant active S6K in the presence or absence of myc-YY2 as a substrate. eIF4B was used as a positive control (n = 3). g Immunoblot analysis of immunoprecipitated (IP) GFP-YY2 and the whole-cell-lysate (WCL) from HeLa cells expressing GFP-YY2 (n = 3). h Immunoblot analysis of immunoprecipitated GFP-YY2 and the WCL from HeLa cells expressing GFP-YY2 (n = 3). i The evolutionarily conserved putative S6K phosphorylation site in YY2. j Immunoblot analysis of immunoprecipitated GFP-YY2 and the WCL from HeLa cells stably expressing wild-type (WT) GFP-YY2 or mutant (T336A) GFP-YY2, treated with 40 μM PF-4708671 for 1 hr (n = 3). k Immunoblot analysis of immunoprecipitated GFP-YY2 and the WCL from HeLa cells expressing WT GFP-YY2 or T336A GFP-YY2 (n = 3). l, m Network view of predicted E3 ubiquitin ligase-YY2 interactions by UbiBrowser 2.0. Confidence level of (m). n Decreased EGFP-YY2 expression in HeLa cells transfected with Smurf1-FLAG (n = 3). o Decreased Smurf1 ubiquitination in HeLa cells transfected with S6K. Immunoblot analysis of GFP-immunoprecipitates from EGFP-YY2 HeLa cells transfected with Smurf1-FLAG or HA-WT-S6K for 24 hr (n = 3). p Immunoblot analysis of GFP-and IgG control immunoprecipitates from EGFP-YY2 HeLa cells transfected with Smurf1-FLAG (n = 3). q Immunoblot analysis of B7-H3 expression in Tsc2−/− 105K cells, transfected with control or myc-YY2 for 48 hr and treated with 20 nM rapamycin or vehicle for the last 24 hr (n = 3). Source data is provided in the Source data file.