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. 2022 Nov 28;30(2):369–382. doi: 10.1038/s41418-022-01089-7

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© The Author(s) 2022

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 — A, B Western blots and quantification for comparing the protein level of SPY1 in the cells of EV, WT, and hSOD1^G93A (n = 3). C The relative mRNA level of SPY1 was assessed by qRT-PCR (n = 6). D, E MG132 (25 μM) was added into hSOD1^G93A and WT cells for 6 h to evaluate the effect on expression of SPY1 (n = 3). F After transfection with SPY1-Flag for 24 h and incubation with MG132 (10 μM) for 4 h in cells of hSOD1^G93A and WT, the supernatants of cell lysates were immunoprecipitated with anti-Flag and immunoblotted with anti-ubiquitin. G After transfection with SPY1-Flag and SKP2-HA for 24 h, the supernatants of cell lysates were immunoprecipitated with Flag (left) or HA (right) antibodies and immunoblotted with HA (lower panel) or Flag (upper panel) antibodies. H UbiBrowser was used to predict E3 ligase interacted with SPY1 as substrate. The prediction score of interaction was expressed from high to low by connecting lines from thick to fine. I, J Western blots and quantification for changes of SPY1 in NSC34 cells transfected with different doses of MDM2-HA (n = 3). K Alteration in ubiquitination of SPY1 with the increase of transfected MDM2. IP, anti-Flag. IB, anti-ubiquitin. L After transfection with SPY1-Flag and MDM2-HA for 24 h, immunoprecipitated with Flag (left) or HA (right) antibodies and immunoblotted with HA (lower panel) or Flag (upper panel) antibodies. M, N Western blots and quantification for NEDD4, SKP2, and MDM2 in the cells of EV, WT, and hSOD1^G93A (n = 3). Values represent mean ± SD. Statistical analysis by one-way ANOVA followed by Tukey’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001.