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. 2024 Jan 29;43(5):754–779. doi: 10.1038/s44318-024-00036-1

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© The Author(s) 2024

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/. Creative Commons Public Domain Dedication waiver http://creativecommons.org/publicdomain/zero/1.0/ applies to the data associated with this article, unless otherwise stated in a credit line to the data, but does not extend to the graphical or creative elements of illustrations, charts, or figures. This waiver removes legal barriers to the re-use and mining of research data. According to standard scholarly practice, it is recommended to provide appropriate citation and attribution whenever technically possible.

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(A) Schematic representation of the process to select monobodies against OPTN using TRAP display. The BC, CD, and FG loops in the monobody structure (PDB: 1TTG) are shown in orange. (B) NDP52 KO HeLa cells expressing Parkin, mt-Keima, and YFP or YFP-tagged monobodies (MonoB) were treated with antimycin A and oligomycin (AO) for 5 h and analyzed by FACS. Representative FACS data with the percentage of cells exhibiting lysosomal positive mt-Keima are shown. (C) Mitophagy rate (percentage of cells having lysosomal positive Keima) in (B) were quantified. Error bars represent mean ± s.d. of four independent experiments. (D) Mitophagy rate (percentage of cells having lysosomal positive Keima) using OPTN KO HeLa cells were quantified. Error bars represent mean ± s.d. of four independent experiments. (E) NDP52 KO HeLa cells expressing Parkin and YFP or YFP-tagged MonoB were analyzed by immunoblotting (IB). (F) The cells in (E) were co-immunoprecipitated (co-IP) using GFP-Trap. The bound fractions were analyzed by IB. (G) Recombinant GST-OPTN (26–196 aa, 26–119 aa, or 133–196 aa) were incubated with recombinant MonoB. The glutathione sepharose bound fractions were analyzed by CBB staining. (H) Recombinant GST-TBK1 (677–729 aa) and MonoB were incubated with either recombinant GFP or OPTN (26–196 aa)-GFP. GST-TBK1 (677–729 aa) was pulled down with glutathione sepharose and the bound fractions were analyzed by CBB staining. (I) The levels of OPTN (26–196 aa)-GFP pulled down with GST-TBK1 (677–729 aa) in (H) were quantified. The level of OPTN (26–196 aa)-GFP pulled down without MonoB was set to 100. Error bars represent mean ± s.d. of three independent experiments. Data information: n.s. not significant, *P < 0.05, ***P < 0.001 by two-tailed Dunnett’s test (C,D,I). Source data are available online for this figure.