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. 2017 Jul 27;7:6737. doi: 10.1038/s41598-017-06934-1

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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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Interaction of NS3 with TRAF6. (a) Interaction of NS3 with TRAF6 in a yeast two-hybrid system. The yeast strain Y2HGold was cotransformed with the prey plasmid AD-TRAF6 and the bait plasmid BD or BD-NS3. Cotransformation with BD/AD, BD-Lamin/AD-T, and BD-p53/AD-T were used as a blank, negative and positive controls, respectively. (b) Exogenous co-IP analysis of NS3 and TRAF6 in PAMs. Cells were co-transfected with plasmids NS3-GFP and TRAF6-Flag. PAMs co-transfected with NS3-GFP and RFP-Flag were used as negative controls. A quarter of the cell extract was subjected to the input assay to assess β-actin, Flag-fusion, and GFP-fusion protein levels. The rest of the extract was subjected to IP assay. Western blot detected proteins with a mouse anti-GFP mAb and a rabbit anti-Flag pAb. (c) Endogenous co-IP analysis of NS3 and TRAF6 in PAMs. Cells were transfected with plasmid NS3-Flag, and RFP-Flag-transfected PAMs were used as negative controls. The input assay was performed using a quarter of the cell extract to assess β-actin, Flag fusion protein, and TRAF6 levels. The precipitated proteins were detected with a rabbit anti-TRAF6 pAb and a rabbit anti-Flag pAb. (d) GST-NS3 pull-down assay. The GST and GST-NS3 proteins expressed in Escherichia coli Rosetta (DE3) cells were immobilized on a glutathione agarose resin, followed by incubation of the resin with the cell lysates containing TRAF6-Flag protein. After washing, the bound proteins were detected by Western blot using a mouse anti-Flag mAb. The expression of input proteins (TRAF6-Flag, GST or GST-NS3) was confirmed by Western blot using a mouse anti-Flag mAb and a mouse anti-GST mAb, respectively. (e) GST-TRAF6 pull-down assay. The GST and GST-TRAF6 proteins expressed in Escherichia coli Rosetta (DE3) cells were immobilized on the glutathione-agarose resin. The resin conjugated with GST or GST-TRAF6 was incubated with the cell lysates containing NS3-GFP protein. After washing, the eluted proteins were detected by Western blot using a mouse anti-GFP mAb.