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. 2021 May 7;28:35. doi: 10.1186/s12929-021-00731-9

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© The Author(s) 2021

Open AccessThis 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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 3 — NEDD4 mediates mono- and polyubiquitination of IGPR-1. a A schematic of in vitro ubiquitination assay is shown. b In vitro ubiquitination assay was carried out as described in the material and method section using biotinylated ubiquitin (Ub), E2 conjugating enzymes, purified GST-NEDD4 and GST-cytoplasmic domain of IGPR-1. The samples were resolved on SDS-PAGE and ubiquitinated GST-IGPR-1 was detected by Western blotting via Streptavidin-HRP antibody. c–e Coomassie blue staining of GST-IGPR-1, GST-NEDD4 and E2 conjugating enzymes. f HEK-293 cells expressing IGPR-1-Myc were transfected with an empty vector or wild-type NEDD4-HA, cells were lysed and whole cell lysates were immunoprecipitated with an anti-Myc antibody followed by Western blot analysis using anti-ubiquitin (FK2) antibody, K63-poly-ubiqutin antibody or K48-poly-ubiqutin antibody. Whole cell lysates (WCL) were blotted for IGPR-1-Myc and NEDD4-HA