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. 2017 Dec 20;8:2232. doi: 10.1038/s41467-017-01974-7

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© The Author(s) 2017

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 — Cellular assays to test the ubiquitination of E6AP substrates identified by OUT. a Inhibition of E6AP expression in HEK293 cells by shE6AP was confirmed with Western blot probed with an antibody against E6AP. b–g Ubiquitination of MAPK1 b, PRMT5 c, CDK1 d,CDK4 e, β-catenin f and UbxD8 g in HECK293 cells was assayed by immunoprecipitation with antibodies against each substrate proteins and probing the ubiquitination levels of the proteins with an anti-UB antibody on the western blots. After 1.5-h treatment of cells with MG132, ubiquitination of each target protein was compared among the blank HEK293 cell (HEK293), HEK293 expressing shE6AP (shE6AP), HEK293 expressing both shE6AP and recombinant E6AP (shE6AP + OE), and HEK293 expressing recombinant E6AP (OE). h Ubiquitination of HHR23A, a known E6AP substrate, was assayed as a control. Rabbit IgG was used as a control for immunoprecipitation in b and e. Mouse IgG was used as a control for immunoprecipitation in c,d and f–h. All blots are representative of at least three independent experiments