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. 2017 Mar 28;7:477. doi: 10.1038/s41598-017-00490-4

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© The Author(s) 2017

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

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CaREL1 is localized at nucleus and has E3 ligase activity. (a) Subcellular localization of the 35S:CaREL1-GFP fusion protein in Nicotiana benthamiana epidermal cells. The 35S:CaREL1-GFP construct was expressed via agroinfiltration of N. benthamiana leaves and observed using a confocal laser-scanning microscope. DAPI staining was used as a marker for the nucleus. The scale bar represents 20 μm. (b) Auto-ubiquitination of CaREL1. In the presence of ubiquitin, E1 (UbE1), and E2 (UBHPC H5b), maltose-binding protein (MBP)–CaREL1 fusion proteins displayed E3 ubiquitin ligase activity. Detection of MBP–CaREL1 auto-ubiquitination. MBP–CaREL1^C205S/C208S was loaded as negative control. MBP–CaREL1 fusion proteins were detected using MBP and ubiquitin antibodies; shifted bands indicate the attachment of ubiquitin molecules.