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. 2024 Oct 1;56(10):2246–2259. doi: 10.1038/s12276-024-01322-y

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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/.

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Fig. 2 — a A gel stained with Coomassie blue for mass spectrometry analysis. Cell extracts from A10 cells treated with Pi (4 mM) or MLN4924 (1 μM) were immunopurified with an anti-NEDD8 antibody and subjected to SDS‒PAGE. The bands indicated by arrows were then digested with trypsin and analyzed by mass spectrometry. b Cell lysates from Pi-treated A10 cells were immunoprecipitated with anti-NEDD8 and immobilized with anti-PARP-1. PARP-1 neddylation increased in Pi-induced VC. c Whole-cell lysates from 293 T cells transfected with the indicated constructs were subjected to Ni-NTA pull-down. PARP-1 neddylation was blunted in NEDD8-ΔGG compared with that in NEDD8 WT. d A PLA was performed. NEDD8-conjugated PARP-1 is dissociated by MLN4924 (1 μM). Scale bar=20 μM. e Immunoprecipitation with anti-PARP-1. Pi-induced PARP-1 neddylation is attenuated by MLN4924 (1 μM) in A10 cells. PARP-1 activity was measured by detecting the poly(ADP)-ribosylation (PAR) polymer (f) and conducting a PARP-1 enzyme activity assay (g). Pi-induced PARP-1 activity was blunted by MLN4924 treatment in A10 cells. n = 6 per group, independent experiments. Note: “*” indicates a nonspecific band. A.U., arbitrary units. The data are presented as the means ± SEMs. Statistical significance was tested via ANOVA with Tukey’s HSD test.