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A Schematic diagram of the quantitative proteomics analysis workflow for succinylation modification in U2OS cells after 24-hour treatment with 20 μM Etoposide. B LC-MS identification of the total number of peptides and the total number of loci. C Statistical analysis of differentially modified sites after treatment of U2OS cells with 20 μM of Etoposide. D The subcellular localization classification map was performed for the screened differential proteins. E After 20 μM Etoposide processing U2OS enrichment of COG/KOG on differences in modified protein function analysis. F Functional enrichment analysis of differentially modified proteins. KEGG pathway were included. G Mainly in the cell of NADPH metabolic pathways. H – M Pan-succinylated lysine antibody using western blotting. Succinylation levels of G6PD, 6PGD, IDH1, IDH2, MTHFD2, ME2-Flag ectopically expressed in HEK293T cells were measured by the pan-succinylated lysine antibody. N Succinylation levels of ectopically expressed ME2 in HEK293T cells were measured. Cells were treated <t>with</t> <t>3-NPA</t> (3 mM), <t>DES</t> (20 mM) or Glycine (100 mM) were used to treat cells for 24 h. O Succinylation levels of ectopically expressed ME2 in HEK293T cells were measured. Cells were treated with 5 mM NAM for 3 h before harvesting. ME2-Flag was immunoprecipitated from cell lysates and its succinylation was examined with a pan-succinylated lysine antibody.
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A Schematic diagram of the quantitative proteomics analysis workflow for succinylation modification in U2OS cells after 24-hour treatment with 20 μM Etoposide. B LC-MS identification of the total number of peptides and the total number of loci. C Statistical analysis of differentially modified sites after treatment of U2OS cells with 20 μM of Etoposide. D The subcellular localization classification map was performed for the screened differential proteins. E After 20 μM Etoposide processing U2OS enrichment of COG/KOG on differences in modified protein function analysis. F Functional enrichment analysis of differentially modified proteins. KEGG pathway were included. G Mainly in the cell of NADPH metabolic pathways. H – M Pan-succinylated lysine antibody using western blotting. Succinylation levels of G6PD, 6PGD, IDH1, IDH2, MTHFD2, ME2-Flag ectopically expressed in HEK293T cells were measured by the pan-succinylated lysine antibody. N Succinylation levels of ectopically expressed ME2 in HEK293T cells were measured. Cells were treated with 3-NPA (3 mM), DES (20 mM) or Glycine (100 mM) were used to treat cells for 24 h. O Succinylation levels of ectopically expressed ME2 in HEK293T cells were measured. Cells were treated with 5 mM NAM for 3 h before harvesting. ME2-Flag was immunoprecipitated from cell lysates and its succinylation was examined with a pan-succinylated lysine antibody.

Journal: Communications Biology

Article Title: SIRT5-mediated desuccinylation of MTHFD2 enhances chemoresistance in breast cancer cells by reducing therapy-induced senescence

doi: 10.1038/s42003-025-08878-z

Figure Lengend Snippet: A Schematic diagram of the quantitative proteomics analysis workflow for succinylation modification in U2OS cells after 24-hour treatment with 20 μM Etoposide. B LC-MS identification of the total number of peptides and the total number of loci. C Statistical analysis of differentially modified sites after treatment of U2OS cells with 20 μM of Etoposide. D The subcellular localization classification map was performed for the screened differential proteins. E After 20 μM Etoposide processing U2OS enrichment of COG/KOG on differences in modified protein function analysis. F Functional enrichment analysis of differentially modified proteins. KEGG pathway were included. G Mainly in the cell of NADPH metabolic pathways. H – M Pan-succinylated lysine antibody using western blotting. Succinylation levels of G6PD, 6PGD, IDH1, IDH2, MTHFD2, ME2-Flag ectopically expressed in HEK293T cells were measured by the pan-succinylated lysine antibody. N Succinylation levels of ectopically expressed ME2 in HEK293T cells were measured. Cells were treated with 3-NPA (3 mM), DES (20 mM) or Glycine (100 mM) were used to treat cells for 24 h. O Succinylation levels of ectopically expressed ME2 in HEK293T cells were measured. Cells were treated with 5 mM NAM for 3 h before harvesting. ME2-Flag was immunoprecipitated from cell lysates and its succinylation was examined with a pan-succinylated lysine antibody.

Article Snippet: DES (HY-44134), DMK (HY-Y0836), 3-NPA (HY-W012875) were purchased from MCE.

Techniques: Quantitative Proteomics, Modification, Liquid Chromatography with Mass Spectroscopy, Functional Assay, Western Blot, Immunoprecipitation