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. 2018 Feb 21;9(3):302. doi: 10.1038/s41419-018-0370-y

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© The Author(s) 2018

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. 7 — a Model of WASH, Arp2/3, E2, E3, and K63 ubiquitin regulation on F-actin polymerization. b Immunoblotting of WASH total Ubiquitin in cotransfected HEK293T cells. HEK293T cells were cotransfected with YFP-WASH and with (+) or without (−) FLAG-Trim59FL or FLAG-Vector as well as with HA-Ub. Immunoprecipitation was performed by anti-WASH. Trim59 and WASH were detected by anti-FLAG and anti-WASH antibodies. The polyubiquitination of WASH was detected by anti-HA. c Immunoblotting of WASH K63-linked ubiquitin in cotransfected HEK293T cells. HEK293T cells were cotransfected with YFP-WASH and with (+) or without (−) FLAG-Trim59 as well as with HA-Ub, HA-K48-Ub, or HA-K63-Ub. Immunoprecipitation was performed by anti-WASH. Trim59 and WASH were detected by anti-FLAG and anti-WASH antibodies. The polyubiquitination of WASH was detected by anti-HA. d Immunoblotting of WASH K63-linked ubiquitin in cotransfected HEK293T cells. HEK293T cells were cotransfected with YFP-WASH or YFP-K220R and with (+) or without (−) FLAG-Trim59 as well as with HA-K63-Ub. Immunoprecipitation was performed by anti-WASH. Trim59, WASH and WASH variant were detected by anti-FLAG and anti-WASH antibodies. Polyubiquitination of WASH was detected by anti-HA. e Immunoblotting of WASH K63-linked ubiquitin in cotransfected HEK293T cells. HEK293T cells were cotransfected with FLAG-Trim59FL and its fragments (T2 toT5) and with YFP-WASH (+) as well as HA-K63-Ub (+). Immunoprecipitation was performed by anti-WASH. Trim59, Trim59 fragments and WASH were detected by anti-FLAG and anti-WASH antibodies. Polyubiquitination of WASH was detected by anti-HA. f Immunoblotting of total Ub and K63-Ub of endogenous WASH from F1 ESCs after treated with specific Trim59 and Trim27 siRNA.Immunoprecipitation was performed by anti-WASH. Trim59, Trim27, and WASH were detected by anti-Trim59, anti-Trim27, and anti-WASH antibodies. Immunoblot analysis of Ub-WASH (top blot), K63-Ub-WASH (below blot) by anti-Ub and anti- K63 antibodies. g Immunofluorescence assay of F-actin assembly in WASH siRNA or siRNA control transfected F1 ESCs. Scale bar, 10 µm. *P < 0.05, **P < 0.01, ***P < 0.005; Ns no significance. Polyubi. polyubiquitination, IP immunoprecipitation, IB immunoblot assay, Actin a loading control, WCL whole-cell lysates, Ctr.IgG isotype control IgG