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. 2018 Feb 15;9:688. doi: 10.1038/s41467-018-03107-0

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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. 2 — USP35 is a DUB for Aurora B. a HEK293T cells were transfected with HA-Aurora B alone or in combination with Flag-USP35. The interaction between Flag-USP35 and HA-Aurora B was detected by immunoblotting after immunoprecipitation with an anti-Flag antibody. TCL, total cell lysates. b HEK293T cells were transfected with Flag-USP35 alone or in combination with HA-Aurora B. The interaction between Flag-USP35 and HA-Aurora B was detected by immunoblotting after immunoprecipitation with an anti-HA antibody. c The interaction between endogenous USP35 and Aurora B was detected by immunoblotting after immunoprecipitation with an anti-Aurora B antibody. d HEK293T cells transfected with His-ubiquitin alone or in combination with HA-Aurora B, Flag-USP35 or Flag-USP35^C450A were synchronized by a treatment with 100 ng/mL nocodazole (NOC) for 18 h and then treated with the proteasome inhibitor MG132 for 4 h. Aurora B ubiquitination was observed using a Ni-NTA-mediated pulldown assay. e HeLa cells transfected with CONi or USP35i were synchronized in prometaphase by a treatment with 100 ng/mL NOC for 18 h. The cell lysates were immunoblotted using the indicated antibodies. f HeLa cells were transfected with USP35i-#1 or USP35i-#2 alone or in combination with HA-Aurora B. Cells showing several defects were counted after immunofluorescence staining with β-tubulin Cy3 antibody. One-hundred cells per group were examined from three independent experiments. The data in a part f represent the mean ± SD (*P < 0.05; **P < 0.005, t-test)