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. 2020 Feb 25;10:3422. doi: 10.1038/s41598-020-60241-w

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© The Author(s) 2020

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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Ubiquitin-binding activity of ASCC2 is crucial for RQC induction. (A) Top: Schematic drawing of the domain structure of ASCC2. Bottom: Amino-acid sequence alignment of conserved residues in CUE domains of the indicated proteins. Mutated residues in Ub-m are shown in bold red. (B) Recombinant HA-ASCC2 or HA-ASCC2-Ub-m mutant (Input), and GST or GST-Ubiquitin were mixed and reacted in vitro, and then pulled down with Glutathione Sepharose. GST-tagged proteins were eluted with glutathione (elution). Both input and elution fractions were analyzed by western blotting with antibodies against HA. Elution fractions were also analyzed by CBB staining. (C) HA-ASCC2 or HA-ASCC2-Ub-m mutant was co-transfected into HEK293T cells along with FLAG-ASCC3, and immunoprecipitated with anti-FLAG antibody beads. FLAG-tagged proteins were eluted with FLAG peptides, and the eluted fraction was analyzed by western blotting with antibodies against FLAG and HA. (D) HA-ASCC2 or HA-ASCC2-Ub-m mutant was co-transfected into ASCC2 KD cells along with the V5-GFP-K(AAA)₂₄-FLAG-HIS3 reporter, and protein was analyzed by western blotting with anti-V5 antibodies. Arrest products highlighted by the red dashed line are shown with a longer exposure (V5 enhanced). (B–D) Cropped blots are displayed; full uncropped blots are available in Supplemental Fig. S8. Blots shown are representative of three independent experiments.