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. 2021 Apr 1;11:7320. doi: 10.1038/s41598-021-86847-2

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© The Author(s) 2021

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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Effect of inhibitors of the proteasomal and autophagic-lysosomal pathways on the degradation of human wild type DJ-1 and missense mutants transfected in DJ-1-null MEFs. DJ-1-null MEFs were transiently transfected with the indicated untagged human DJ-1 (hDJ-1) constructs and 48 h after transfection were kept in complete medium (C) or treated with 25 µg/mL cycloheximide (CHX) in the absence (DMSO) or the presence of 10 µM MG-132, 20 mM NH₄Cl, 20 mM NH₄Cl plus 50 µM leupeptin (Leu) or 10 mM 3-methyl adenine (3MA) plus 5 µM E64 for 12 or 24 h, as indicated. Total cell lysates were analysed by Western and immunoblot with the corresponding specific antibodies. (A) Panels show representative immunoblots with anti-DJ-1 polyclonal antibody of human wild type DJ-1 (WT), M26I, A107P, E163K and L172Q. (B) Panels show the results obtained with transfected hDJ-1 L10P, P158Δ and L166P developed with anti-DJ-1 polyclonal antibody. Anti-tubulin antibodies were used as total protein loading control. (C) Graphs below each panel show the quantifications of the levels of DJ-1 protein respect to untreated cells as control. Values are expressed as mean ± s.e.m from three different experiments. Significant differences were found for hDJ-1 A107P between control cells and cells treated with CHX (**p = 0.002), CHX in combination with MG-132 (**p = 0.004), CHX in combination with NH₄Cl (**p = 0.005), CHX in combination with NH₄Cl plus leupeptin (**p = 0.003) and CHX in combination with 3MA plus E64 (**p = 0.001); for hDJ-1 E163K between control cells and cells treated with CHX (*p = 0.01), CHX in combination with MG-132 (*p = 0.03), CHX in combination with NH₄Cl (*p = 0.02), CHX in combination with NH₄Cl plus leupeptin (*p = 0.01) and CHX in combination with 3MA plus E64 (*p = 0.03); for hDJ-1 L172Q between control cells and cells treated with CHX (*p = 0.01), CHX in combination with MG-132 (*p = 0.03), CHX in combination with NH₄Cl (*p = 0.04), CHX in combination with NH₄Cl plus leupeptin (*p = 0.04) and CHX in combination with 3MA plus E64 (*p = 0.04); for hDJ-1 L10P between control cells and cells treated with CHX (**p = 0.0001), CHX in combination with MG-132 (**p = 6E−05), CHX in combination with NH₄Cl (**p = 0.0003), CHX in combination with NH₄Cl plus leupeptin (**p = 9E−05) and CHX in combination with 3MA plus E64 (**p = 0.0002); for hDJ-1 P158Δ between control cells and cells treated with CHX (**p = 5E−06), CHX in combination with MG-132 (**p = 9E−06), CHX in combination with NH₄Cl (**p = 1E−05), CHX in combination with NH₄Cl plus leupeptin (**p = 1E−05) and CHX in combination with 3MA plus E64 (**p = 4E−07) and for hDJ-1 L166P between control cells and cells treated with CHX (**p = 3E−06), CHX in combination with MG-132 (**p = 2E−05), CHX in combination with NH₄Cl (**p = 5E−05), CHX in combination with NH₄Cl plus leupeptin (**p = 2E−06) and CHX in combination with 3MA plus E64 (**p = 0.0002). n.s. not significant.