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. 2018 Mar 7;9:976. doi: 10.1038/s41467-018-03339-0

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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. 1 — Itch ubiquitylates and binds SuFu. a, b HEK293T cells were transfected with plasmids expressing HA-ubiquitin (HA-Ub) in the presence of different E3 ubiquitin ligases (a) or increasing amount of Flag-Itch (b). Cell lysates were immunoprecipitated with an anti-SuFu antibody, and ubiquitylated forms were revealed with an anti-HA antibody. c HEK293T cells were co-transfected with Flag-Itch and HA-SuFu as indicated. Interaction between Itch and SuFu was detected by immunoprecipitation (IP) followed by immunoblot (IB) analysis with the indicated antibodies. d Interaction between endogenous Itch and SuFu was detected in HEK293T cells by immunoprecipitation followed by immunoblot analysis with the indicated antibodies. e GST-Itch was bound to glutathione-sepharose beads and used for in vitro pull-down assay. In vitro translated ³⁵S-labelled SuFu was incubated with free GST control or GST-Itch. After GST pull-down, the protein complex was detected by fluorography. Coomassie blue staining shows the expression levels of recombinant proteins GST-Itch or GST only. f GST-SuFu was bound to glutathione-sepharose beads and used for in vitro pull-down assay. Untagged Itch recombinant protein was incubated with free GST control or GST-SuFu. After GST pull-down, the protein–protein interaction was detected by IB with an anti-Itch antibody. Coomassie blue staining shows the expression levels of recombinant proteins GST-SuFu or GST only. g Schematic representation of Itch and its interaction with SuFu. h, i GST-HECT (h) or GST-4WWs (i) were bound to glutathione-sepharose beads and used for in vitro pull-down assay with in vitro translated ³⁵S-labelled SuFu. After GST pull-down, protein complexes were analysed by IB. j A GST pull-down assay with GST-WW1, -WW2, -WW3, or -WW4 and in vitro translated ³⁵S-labelled SuFu was carried out as described in e