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. 2017 Jun 6;8:15662. doi: 10.1038/ncomms15662

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Copyright © 2017, The Author(s)

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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(a) Western blot analysis of NANOS2 immunoprecipitated by an anti-MYC (NEDD4) antibody in HEK 293T cells. (b) Western blot analysis of MBP-NANOS2 purified with either GST or GST-NEDD4. Yellow arrowheads indicate nonspecific bands (n=3). (c) A diagram of the method used for the in situ analysis of the interaction between NEDD4 and NANOS2 by means of a bimolecular fluorescent complementation assay (BiFC). CTV: C terminus of VENUS protein, NTV: N-terminus of VENUS protein. (d–g) Analysis of NIH3T3 cells transfected with FLAG-NANOS2-CTV and MYC-NEDD4-NTV. Interaction between NANOS2 and NEDD4 was visualized by VENUS signals. The NANOS2 and NEDD4 proteins were immunostained as red and magenta, respectively (d). Dcp1a (P body marker) staining was colocalized with NANOS2-only foci but not NANOS2-NEDD4 double-positive foci (e). Open triangles indicate NANOS2 single-positive foci. Filled triangles indicate NANOS2-NEDD4 double-positive foci, which are not colocalized with Dcp1a. Scale bar, 5 μm. (f,g) The interaction of NEDD4 and NANOS2 (VENUS) was observed in both endosomes (f, TfR) and lysosomes (g, LAMP1). Three independent transfections were performed. Scale bar, 5 μm. (h) Western blot analysis showing that NEDD4 directly promotes MBP-NANOS2 ubiquitination in vitro. (i) Western blot analysis of 293T cells cotransfected with plasmids expressing HA-tagged ubiquitin, FLAG-tagged NANOS2, and MYC-tagged NDFIP2 and NEDD4 (n=3). IP was performed with an anti-FLAG (NANOS2) antibody.