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. 2022 Apr 19;13:2089. doi: 10.1038/s41467-022-29502-2

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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. 6 — a Western blot of HeLa cells synchronized by double thymidine block using phospho-specific antibodies. b Western blot of immunoprecipitates from HeLa cells synchronized by double thymidine block and collected at serial times for immunoprecipitation using SKP2 (upper panel), Cyclin B (middle panel), or ID2 (lower panel) antibodies. E47 and HEB are controls for the immunoprecipitation of endogenous ID2. c Loss of CDH1-mediated regulation of SKP2-S64D stability. Western blot of HeLa cells expressing FLAG-SKP2 WT, S64A, or S64D in the presence or absence of CDH1. d Loss of CDH1-mediated regulation of Cyclin B-S126D stability. Western blot of HeLa cells expressing FLAG-Cyclin B WT, S126A, or S126D in the presence or absence of CDH1. e In vivo ubiquitylation of HeLa cells co-expressing FLAG-SKP2 WT or phospho-mutants and HA-ubiquitin. HA western blot of cellular lyasates immunoprecipitated with FLAG antibody. WCL, whole cellular lysates. f In vivo ubiquitylation of HeLa cells co-expressing FLAG-Cyclin B1 WT or phospho-mutants and HA-ubiquitin. HA western blot of cellular lysates immunoprecipitated with FLAG antibody. WCL whole cellular lysates. g Loss of interaction between the SKP2-S64D and core APC is independent of CDH1. FLAG-immunoprecipitation-western blot of HeLa cells expressing FLAG-SKP2 WT or phospho-mutants in the presence or the absence of CDH1 siRNA (left panel). Right panel, whole cellular lysate (WCL). h Loss of interaction between the Cyclin B-S126D and core APC is independent of CDH1. FLAG-immunoprecipitation-western blot of HeLa cells expressing FLAG-Cyclin B WT or phospho-mutants in the presence or the absence of CDH1 siRNA (left panel). Right panel, whole cellular lysate (WCL). Total and phosphorylated proteins are from independent gels; APC1, APC3, CDH1 are from the same gel in each panel; E47, HEB, pHH3, SKP2 and Cyclin B1 are from independent gels; HA and FLAG/Vinculin in panel e are from different gels; Loading controls are from the same gel as FLAG or HEB in panel a. Molecular weight markers are indicated in kDa. Experiments were repeated two times with similar results.