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. 2017 Oct 3;7:12600. doi: 10.1038/s41598-017-12707-7

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© The Author(s) 2017

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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NUMBL overexpression inhibits TRAF6 and NEMO by inducing K48-pUB of TRAF6 and NEMO. (A) WT bone marrow cells expressing pMX-NUMBL showed decreased expression of TRAF6 and NEMO upon RANKL stimulation when compared with pMX-GFP expressing cells after 2 days of RANKL stimulation. Plat-E cells were co-transfected with fixed amount of pMX-NUMBL in the presence of increasing amount of (B; left panel) TAK1, (C; left panel) TRAF6 and (D; left panel) NEMO or with increasing amount of pMX-NUMBL with fixed amount of (B; right panel) TAK1, (C; middle panel) TRAF6 and (D; middle panel) NEMO. 2 days after transfection the cells were lysed and immuno-probed with indicated antibodies. Immuno-blots indicate that increasing NUMBL expression results in decrease in TAK1, TRAF6 and NEMO expression in Plat-E cells. The expression of (C: right panel) TRAF6 and (D; right panel) NEMO was quantitated using image J. (E) Plat-E cells were co-transfected with NUMBL and TRAF6. Immuno-blot from total cell lysates and TRAF6-immunoprecipitates shows that NUMBL interacts with TRAF6 and leads to its K48-poly-ubiquitine mediated proteasomal degradation. (F) Plat-E cells were co-transfected with NUMBL and NEMO. Immuno-blot from total cell lysates and NEMO-immunoprecipitates shows that NUMBL interacts with TRAF6 and leads to its K48-poly-ubiquitine mediated proteasomal degradation. Full-length Western blots images are included in supplementary data as Figure S5.A-F.