anti impdh2  (Proteintech)

Journal: Redox Biology

Article Title: Alpha-ketoglutarate accelerates granulocyte-monocyte progenitor differentiation and atherosclerotic plaque inflammation via oxoglutarate receptor 1

doi: 10.1016/j.redox.2026.104134

Figure Lengend Snippet: PNP promoted GMP proliferation and enhanced myeloid cell production via inosine-5′-monophosphate dehydrogenase 2 (IMPDH2). Violin plot presenting PNP expression in GMPs and subpopulations by scRNA-seq analysis. ( A–F ) BMCs were exposed to PNP (0–1 U/mL) for 24 and 72 h. FACS analysis of the frequency of HSPCs, GMPs, and Pro-NEU. ( G–I ) After 3 days of PNP treatment, the proportion of neutrophils and monocytes in BMCs was quantified by FACS. ( J–Q ) Lineage −/low cells were treated with 0 or 0.1 U/mL PNP for 24 h. Ribose-5-phosphate and IMPDH2 expression was measured by ELISA. BMCs were treated with PNP in the absence or presence of IMPDH2 inhibitor for 24 h. EDU + GMPs were analyzed by FACS. In parallel, the frequency of HSPCs, GMP, Pro-NEU, and cMOP was analyzed by FACS. ( R–S ) Lineage −/low cells were treated with 0 or 0.1 U/mL PNP for 72 h. Neutrophil and monocyte production were evaluated by FACS. BMC: bone marrow cell; ELISA: enzyme-linked immunosorbent assay; FACS: fluorescence-activated cell sorting; GMP: granulocyte-monocyte progenitor; HSPC: hematopoietic stem/progenitor cell; Pro-NEU, Pro-neutrophils; cMOP, common monocyte progenitors; IMPDH2: inosine-5′-monophosphate dehydrogenase 2; PNP: purine nucleoside phosphorylase; scRNA-seq: single-cell RNA sequencing. Two experimental groups were compared using the unpaired two-tailed Student's t-test. In the experiments involving ≥3 groups, one-way analysis of variance followed by Dunnett's test was used.

Article Snippet: Protein was extracted from PNP-treated lineage −/low cells to determine the expression of ribose-5-phosphate, IMPDH2, NAD, SOD, and ATP by ELISA, following the manufacturer's instructions (mouse IMPDH2 ELISA kit [#ml601123], mouse NAD ELISA kit [# JK691233 ], Enzyme-linked Biotechnology, China; mouse SOD ELISA kit [#CSB- E08556 m], Huamei Bioengineering, China; ATP Assay Kit [#HY-K0314], MedChemExpress, US).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Fluorescence, FACS, Single Cell, RNA Sequencing, Two Tailed Test

Journal: Redox Biology

Article Title: Alpha-ketoglutarate accelerates granulocyte-monocyte progenitor differentiation and atherosclerotic plaque inflammation via oxoglutarate receptor 1

doi: 10.1016/j.redox.2026.104134

Figure Lengend Snippet: Correlation of LDL-c and TCA metabolites in human plasma samples. (A-B) The fasting plasma levels of LDL-c and isocitrate were determined in healthy human subjects (n = 40) and their correlation was analyzed by Spearman's correlation analysis. ATP: adenosine triphosphate; ELISA: enzyme-linked immunosorbent assay; GPX: glutathione peroxidase; LDL-c: low-density lipoprotein cholesterol; NAD: nicotinamide adenine dinucleotide; PNP: purine nucleoside phosphorylase; ROS: reactive oxygen species; SOD: superoxide dismutase. (C) A proposed model of HFD-induced skewed myelopoiesis and atherosclerosis via α-ketoglutarate. In mice fed a HFD, isocitrate was elevated and LDL-c increased isocitrate dehydrogenase expression, leading to enhanced α-ketoglutarate production. Acting through OXGR1, α-ketoglutarate upregulated PNP mRNA and protein expression. PNP catabolized guanosine/deoxyguanosine and inosine/deoxyinosine, generating ribose-1-phosphate. By interconversion, ribose-5-phosphate production was increased and initiated de novo purine biosynthesis, resulting in IMPDH2 production and GMP proliferation. PNP metabolized nicotinamide riboside to Nam, decreasing NMN and NAD production. PNP increased NAD kinase expression to further accelerate NAD consumption. Furthermore, PNP increased inflammatory protein expression such as NF-κB. All of which reduced NAD production promoted ROS production and inflammation to facilitate GMP differentiation. Ultimately, the pronounced GMP proliferation and differentiation led to inflammatory myeloid cell production and atherosclerotic progression. GMP: granulocyte-monocyte progenitor; HFD: high-fat diet; IMPDH2: inosine-5′-monophosphate dehydrogenase 2; LDL-c: low-density lipoprotein cholesterol; NAD: nicotinamide adenine dinucleotide; OXGR1: oxoglutarate receptor 1; PNP: purine nucleoside phosphorylase; ROS: reactive oxygen species.

Article Snippet: Protein was extracted from PNP-treated lineage −/low cells to determine the expression of ribose-5-phosphate, IMPDH2, NAD, SOD, and ATP by ELISA, following the manufacturer's instructions (mouse IMPDH2 ELISA kit [#ml601123], mouse NAD ELISA kit [# JK691233 ], Enzyme-linked Biotechnology, China; mouse SOD ELISA kit [#CSB- E08556 m], Huamei Bioengineering, China; ATP Assay Kit [#HY-K0314], MedChemExpress, US).

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Cell Death & Disease

Article Title: USP5 regulates purine metabolism and represents a therapeutic target in esophageal cancer

doi: 10.1038/s41419-026-08683-4

Figure Lengend Snippet: A MS analysis identified proteins commonly enriched in KYSE450 and KYSE30 cells. B , C Endogenous and exogenous co-IP confirmed the interaction between USP5 and IMPDH2. D Molecular docking of USP5 with IMPDH2. E IMPDH2 protein levels were assessed by WB after USP5 knockdown. F IMPDH2 protein stability was assessed by WB after USP5 overexpression, and CHX treatment at various time points. G KYSE150 cells were treated with 3-MA or MG132 after USP5 knockdown, and IMPDH2 stability was analyzed by WB after CHX treatment. H Flag-USP5 WT or Flag-USP5 C335A plasmids were transfected into HEK293 cells, and IMPDH2 ubiquitination was assessed by Western blot after 6 h of MG132 treatment. I In HEK293 cells, Myc-IMPDH2, Myc-IMPDH2-K109R, Myc-IMPDH2-K124R, Myc-IMPDH2-K167R, Myc-IMPDH2-K375R, Myc-IMPDH2-K438R, Myc-IMPDH2-K489R plasmids were transfected, followed by MG132 treatment, and IMPDH2 ubiquitination levels were assessed by Western blot. J Endogenous and exogenous co-IP with SYVN1 and IMPDH2 antibodies was performed by WB in KYSE150 and KYSE450 cells. K SYVN1 plasmid was transfected into HEK293 cells, followed by MG132 treatment, and IMPDH2 ubiquitination levels were assessed by Western blot.

Article Snippet: Primary antibodies included rabbit monoclonal antibodies against IMPDH2 and USP5, and a murine monoclonal antibody against Ki67 (1:400; GB121141 ; Servicebio Technology).

Techniques: Co-Immunoprecipitation Assay, Knockdown, Over Expression, Transfection, Ubiquitin Proteomics, Western Blot, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: USP5 regulates purine metabolism and represents a therapeutic target in esophageal cancer

doi: 10.1038/s41419-026-08683-4

Figure Lengend Snippet: A Measurement of guanine levels in KYSE410 cells following IMPDH2 overexpression. B Measurement of guanine levels in KYSE450 and KYSE150 cells after IMPDH2 knockdown. C Guanine levels in KYSE450 cells after treatment with MPA (mycophenolic acid). D , E MTT and foci formation assays in KYSE150 and KYSE450 cells treated with MPA and supplemented with exogenous guanine. F MTT assay was conducted to measure the proliferation in KYSE150 and KYSE450 cells after IMPDH2 knockdown with exogenous guanine supplementation. G – I CDX model combining IMPDH2 knockdown and free-purine diet; tumor weight ( H ) and volume ( I ) measurements. J In KYSE150 and KYSE450 cells with IMPDH2 knockdown, exogenous guanine was added, and guanine levels were measured. K Western blot validation of USP5 knockdown and IMPDH2 overexpression in KYSE70 cells. L MTT assay evaluating the effect of USP5 knockdown and IMPDH2 overexpression on cell proliferation in KYSE70 cells. M – P . Foci formation and soft agar assays assessing the impact of USP5 knockdown and IMPDH2 overexpression on clonogenic capacity of ESCC cells. Q In USP5-knockdown KYSE150 and KYSE450 cells, Myc-IMPDH2 plasmid was transfected, and guanine levels were measured. R In USP5-knockdown KYSE150 and KYSE450 cells, Flag-USP5, Myc-IMPDH2, and Myc-IMPDH2-K489R plasmids were transfected, followed by MG132 treatment, and guanine levels were measured. S In KYSE150 and KYSE450 cells, Flag-SYVN1 and Myc-IMPDH2 plasmids were transfected, followed by MG132 treatment, and guanine levels were measured. Data are presented as mean ± SD. Statistical significance was assessed by unpaired two-tailed Student’s t test ( A , C , R , S ) two-way ANOVA ( L ) or one-way ANOVA ( B , D , E , F , H , I , J , O , P , Q ) as appropriate. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Primary antibodies included rabbit monoclonal antibodies against IMPDH2 and USP5, and a murine monoclonal antibody against Ki67 (1:400; GB121141 ; Servicebio Technology).

Techniques: Over Expression, Knockdown, MTT Assay, Western Blot, Biomarker Discovery, Plasmid Preparation, Transfection, Two Tailed Test

Journal: Cell Death & Disease

Article Title: USP5 regulates purine metabolism and represents a therapeutic target in esophageal cancer

doi: 10.1038/s41419-026-08683-4

Figure Lengend Snippet: A Western blot analysis of USP5 and IMPDH2 protein expression after MDZ treatment. MDZ represents Mebendazole. B Guanine levels were measured in KYSE150 and KYSE450 cells after 48 hours of MDZ treatment. C USP5 protein stability was assessed in KYSE150 and KYSE450 cells after MDZ treatment followed by CHX treatment. D In KYSE70 and KYSE30 cells, MDZ treatment followed by MG132 treatment was performed, and IMPDH2 ubiquitination levels were assessed by Western blot. E In KYSE450 and KYSE30 cells, SYVN1 was transfected, followed by MDZ (Mebendazole) treatment and MG132 treatment, and IMPDH2 ubiquitination levels were assessed by Western blot. F Immunofluorescence staining was performed to detect USP5 protein levels after Mebendazole treatment. G MTT assay was performed to assess cell proliferation levels of KYSE30, KYSE450, KYSE150, and KYSE510 cells after MDZ treatment. H , I Foci formation and soft agar assays were performed to assess the colony formation ability of KYSE30, KYSE450, KYSE150, and KYSE510 cells after MDZ treatment. J Tumor images from the PDX model after intragastric administration of MDZ. K , L Tumor weight and tumor volume after MDZ (Mebendazole) treatment. Data are presented as mean ± SD. Statistical significance was assessed by two-way ANOVA ( G , K ) or one-way ANOVA ( B , H , I , L ) as appropriate. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Primary antibodies included rabbit monoclonal antibodies against IMPDH2 and USP5, and a murine monoclonal antibody against Ki67 (1:400; GB121141 ; Servicebio Technology).

Techniques: Western Blot, Expressing, Ubiquitin Proteomics, Transfection, Immunofluorescence, Staining, MTT Assay

Journal: Cell Death & Disease

Article Title: USP5 regulates purine metabolism and represents a therapeutic target in esophageal cancer

doi: 10.1038/s41419-026-08683-4

Figure Lengend Snippet: A MTT assay was performed to assess the proliferation of KYSE150 and KYSE450 cells after combined treatment with MDZ and Oxa. Oxa represents Oxaliplatin. B The sensitivity to Oxa was assessed in KYSE150 and KYSE450 cells after USP5 or IMPDH2 knockdown. C . MTT assay was assessed in KYSE450 and KYSE510 cells treated with a combination of MDZ and Oxa. D MTT assay was assessed in KYSE450 and KYSE510 cells treated with a combination of USP5 knockdown and Oxa. E – G The PDX model was used to evaluate the combined treatment of MDZ (25 mg/kg) and Oxa (5 mg/kg) ( E ), with tumor weight ( F ) and tumor volume ( G ) measured and analyzed. H Schematic diagram of the mechanism by which USP5 stabilizes IMPDH2 expression and promotes guanine biosynthesis. The USP5-targeting agent mebendazole enhances oxaliplatin sensitivity when used in combination therapy. Data are presented as mean ± SD. Statistical significance was assessed by two-way ANOVA ( A , G ) or one-way ANOVA ( B , C , D , F ) as appropriate. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Primary antibodies included rabbit monoclonal antibodies against IMPDH2 and USP5, and a murine monoclonal antibody against Ki67 (1:400; GB121141 ; Servicebio Technology).

Techniques: MTT Assay, Knockdown, Expressing