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. 2018 Jul 19;26(5):860–876. doi: 10.1038/s41418-018-0164-x

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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. 4 — Kenny is modified by mixed K63-Ub and M1-Ub chains. a Drosophila S2 cells were transfected with empty vector, HA-tagged Kenny and V5-tagged wild-type LUBEL RBR-LDD. HA immunoprecipitations were performed at denaturing conditions and the samples were subjected to ubiquitin chain restriction (UbiCRest) with OTULIN and vOTU. Samples were analysed by Western blotting with α-M1, α-K63 and α-HA antibodies, n = 2. b Drosophila S2 cells were transfected with DIAP2, His-tagged K63-only Ub and V5-tagged Kenny. His-Ub pulldowns were performed at denaturing conditions and the samples were analysed by Western blotting with α-V5, α-DIAP2 and α-Actin antibodies, n = 3. c Drosophila S2 cells were transfected with empty vector, DIAP2, V5-tagged wild-type or C2704A LUBEL RBR-LDD, and HA-tagged Kenny. HA immunoprecipitations were performed at denaturing conditions and the samples were analysed by Western blotting with α-M1, α-K63, α-V5, α-HA, α-DIAP2 and α-Actin antibodies, n = 3. d Drosophila S2 cells were transfected with empty vector, HA-tagged Kenny and V5-tagged wild-type LUBEL RBR-LDD. M1-Ub chains were isolated from cell lysates with GST-NEMO-UBAN at denaturing conditions and the samples were subjected to UbiCRest with OTULIN and AMSH. Samples were analysed by Western blotting with α-M1 and α-HA antibodies, n = 3