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. 2020 Mar 6;11:1251. doi: 10.1038/s41467-020-15109-y

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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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Fig. 6 — a The scheme of BioID assay for identifying the interaction proteins of DJ-1. b The results of BioID assay. The numbers indicate the proteins identified from each group by mass spectrometry, and the top ten proteins specifically identified in biotin-treated DJ-1-BirA* samples were listed. c The interaction between AHCYL1 (the left view) or AHCYL2 (the right view), and DJ-1 in BirA* system was analyzed. HEK293T cells were transfected with indicated plasmids and treated with DMSO or 50 μM biotin for 24 h before harvested. Input and samples of IP-streptavidin were separated on SDS–PAGE gels and subjected to immunoblotting with anti-Flag and anti-DJ-1 antibodies. Independent experiments are repeated three times and representative data are shown. d The interaction between AHCYL1 and SAHH under different DJ-1 level was analyzed. Indicated HEK293T cells stably expressing AHCYL1 and SAHH were harvested for immunoprecipitation and subjected to immunoblotting with anti-Flag and anti-HA antibodies (#1 sequence of DJ-1 KD was used here). ‘s’ represents short exposure; ‘l’ represents long exposure. Independent experiments are repeated three times and representative data are shown. e Western blot analysis of DJ-1 and AHCYL1 expression in indicated H1299 cells (#1 sequence of DJ-1 KD was used here). Independent experiments are repeated three times and representative data are shown. f Indicated DJ-1 KD (#1 sequence) and AHCYL1 KD H1299 cells were treated with erastin (2 μM) for 12 h, and lipid ROS production was assayed. Data shown represent mean ± SD from three independent experiments. Comparisons were made using the two-tailed, unpaired Student’s t-test; *p < 0.05. g Western blot analysis of the fractions from size-exclusion chromatography experiment, showing less tetrameric SAHH complex (fraction 23–28) in DJ-1 KD H1299 cells (#1 sequence of DJ-1 KD was used here). Independent experiments are repeated three times and representative data are shown.