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. 2017 Dec 20;8:2213. doi: 10.1038/s41467-017-02199-4

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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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Fig. 3 — Dim2 interacts with Utp14, an activator of Dhr1 helicase. a 2-hybrid interaction between Dim2 and Utp14. Yeast 2-hybrid analysis of three individual transformants, harboring the indicated bait (BD) and prey (AD) plasmids. Transformants were analyzed by growth on SDC-Leu-Trp (plating efficiency) and SDC-Leu-Trp-Ade plates (strong interaction). SV40 and p53 served as negative controls. The growth was analyzed after 2 days at 30 °C. b Reconstitution of ctDim2-Utp1-Utp14-Dhr1 complexes. The indicated dimeric ctDim2-Utp14 and trimeric ctDim2-Utp1-Utp14 and ctDim2-Utp14-Dhr1 complexes using Chaetomium thermophilum proteins were assembled by co-expression in yeast followed by split-tag tandem affinity-purification. The reconstituted complexes were analyzed via SDS-PAGE (4–12%) and Coomassie staining. c Yeast strains GAL::scDIM2 with integrated scDhr1-FTpA or scUtp14-FTpA were grown in galactose (GAL) medium or shifted for 8 h to glucose (GLU) containing medium before the indicated bait proteins were tandem affinity-purified in two consecutive steps involving the ProtA—in the first and the Flag-tag in the second step. TCA-precipitated Flag eluates were analyzed by SDS-PAGE (4–12%) and Coomassie staining (upper panel) or western blotting (lower panel) using the indicated antibodies. The indicated protein bands were identified via mass spectrometry. d Final eluates derived from scDhr1-FTpA tandem affinity-purification under conditions of Dim2 expression (GAL) and depletion (GLU, 8 h), respectively, were analyzed by sucrose gradient (15–40%) centrifugation. Gradient fractions (1–14) were TCA-precipitated and analyzed by SDS-PAGE (4–12%) and Coomassie staining (upper panels) and western blotting against Dhr1-Flag (lower panels, long and short exposure times are depicted). The experiments were performed at least twice with consistent results. Uncropped images are shown in Supplementary Fig. 9. L load, S molecular weight protein standard, E eluate