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. 2018 Feb 7;9:545. doi: 10.1038/s41467-018-02951-4

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© The Author(s) 2018

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 — The direct interaction between GLUD1 and SIRT5 causes hypoglutarylation of GLUD1. a, b HCT116 (a) and LoVo (b) cells were immunostained for FLAG-SIRT5 WT/H158Y (in green) and GLUD1 (in red); yellow in the merged magnified images (left) indicates the co-localization. Scale bars indicate 20 μm. c Fluorescence intensity of FLAG-SIRT5 WT/H158Y (green line) and GLUD1 (red line) traced along the white line in HCT116 using the line profiling function of the ImageJ software. d, e FLAG-SIRT5 WT/H158Y were immunoprecipitated with anti-FLAG antibody and followed by western blotting with an anti-GLUD1 antibody in HCT116 (d) and LoVo (e) cells. f, g The interaction between endogenous GLUD1 and SIRT5 in HCT116 (f) and LoVo (g) cells. h GST pull-down assays revealed the interaction between SIRT5 and GLUD1. In vitro-translated SIRT5 was incubated GST fusion of GLUD1 or GST, and analyzed by western blotting. The bound SIRT5 is indicated by an arrow on the right. i, j Exogenous GLUD1 proteins were purified in HCT116 (i) and LoVo (j) cells expressing the control vector, SIRT5 WT, and SIRT5 H158Y; and the glutarylation (GluK) levels of GLUD1 were determined by western blotting. Integrated density values were calculated using the ImageJ software. k Exogenous GLUD1 was purified upon SIRT5 knockdown, and the GluK level of GLUD1 was determined. l HA-tagged GLUD1 proteins were purified and incubated with different concentrations of glutaryl-CoA (0, 1, and 2 mM) at 37 °C for 60 min. The GLUD1 activity was determined. Left, representative images (n = 6). Right, quantification of GLUD1 activity. m Wild-type GLUD1, K399R, K503R, and K545R mutants were transfected into HCT116 cells followed by SIRT5 knockdown. The GLUD1 was immunoprecipitated and the level of GluK was determined. n HCT116 cells expressing HA-tagged GLUD1 WT/K545R mutant were treated with or without SIRT5 siRNAs. The GLUD1 activity was measured and normalized against the protein levels. The results in i–n are the mean ± SD of three independent experiments. P values were calculated by ANOVA with Tukey’s test. *P < 0.05, **P < 0.01, ***P < 0.001, N.S. = not significant for the indicated comparison