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Proteintech rabbit polyclonal anti gapdh
Rabbit Polyclonal Anti Gapdh, supplied by Proteintech, used in various techniques. Bioz Stars score: 97/100, based on 6203 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/rabbit+anti+gapdh/GAPDH+Polyclonal+antibody/pmc13011206-161-48-53
Average 97 stars, based on 6203 article reviews

rabbit polyclonal anti gapdh - by Bioz Stars, 2026-09

97/100 stars

Images

Western Blot:

Article Title: DIDS modulates VDAC1 oligomerization to suppress intrinsic apoptosis and attenuates in vitro and in vivo RSV infection
Article Snippet: Four small interfering RNAs (siRNAs) specifically targeted to VDAC1 ( si-VDAC1-1173, si-VDAC1-1280, si-VDAC1-1686, and si-VDAC1-1778 ) and a non-targeting siRNA control ( siRNA-NC ) were designated and synthesized by GenePharma, Shanghai, China, with the sequences provided in . .. The primary antibodies used for Western blotting were as follows: mouse anti-VDAC1 (ab186321, Abcam, Shanghai, China, diluted to 1:1,000), mouse anti-BCL-2 (12789-1-AP, Proteintech, Wuhan, China, 1:5,000), rabbit anti-GAPDH (60004-1-Ig, Proteintech, Wuhan, China, 1:5,000), and rabbit anti-β-actin (AC048, ABclonal, Wuhan, China, 1:10,000). .. For secondary antibodies in Western blotting, HRP-conjugated goat anti-rabbit IgG (GB23303-1, Servicebio, Wuhan, China, 1:5,000) and HRP-conjugated goat anti-mouse IgG (SA00001-1, Proteintech, Wuhan, China, 1:3,000) were utilized.

Article Title: Discovery of FLC-8 as the First Covalent FLT3 Inhibitor Targeting Cys807 for FLT3 Mutant Acute Myeloid Leukemia.
Article Snippet: FLT3 is a validated therapeutic target in acute myeloid leukemia (AML), yet resistance mutations frequently limit current inhibitors.. Here, we report a series of 6-methylisoxazolo[5,4-b]pyridin-3-amines that covalently target Cys807, a previously unexploited nucleophilic residue within the FLT3 kinase domain.. Compound 18 (FLC-8) potently inhibited FLT3-WT (IC50 = 10.2 nM) and clinically relevant mutants G697R (IC50 = 11.6 nM) and N676D (IC50 = 24.1 nM).

Article Title: Fluorescence polarization-based discovery of natural RORγ orthosteric inhibitors.
Article Snippet: Retinoic acid receptor-related orphan receptor γ (RORγ) is a key transcriptional regulator of the T helper 17 cell and has emerged as a promising therapeutic target for autoimmune diseases.. To identify novel orthosteric inhibitors of RORγ, we designed and synthesized ten fluorescent probes targeting the RORγ orthosteric site.. Among them, probe 19g exhibited the best performance (Kd = 252 nM, fluorescence quantum yield = 29.7%) and was subsequently employed to establish a fluorescence polarization (FP)-based assay for screening natural orthosteric binders.

Membrane:

Article Title: Integrating GlycoSHIELD Modeling and DNA-PAINT SMLM to Map the Glycosylation-Dependent Distribution of the Na,K-ATPase
Article Snippet: Electrophoresis was performed for 45 minutes at 150 V and transferred onto a membrane using a Turbo transfer system (Biorad, 25 A, 25 V, 3 min). .. The membrane was blocked for 1 hour at room temperature using 5% BSA in TBS-T (0.2% Tween) and incubated overnight at 4°C with rabbit anti-GFP (1:1000, CAB4211, ThermoFischer) and rabbit anti-GAPDH (1:1000, 10494-1-AP, Proteintech) primary antibodies. .. After three 5-minute washes in TBS-T, the membrane was incubated with anti-Rabbit IRDye 680RD (1:3000, 926-68071, Licor) in 5% BSA TBS Tween for 1 hr at room temperature with mild agitation.

Incubation:

Article Title: Organelle stress in pyometra: Molecular evidence of endoplasmic reticulum stress and mitochondrial dysfunction.
Article Snippet: This study aimed to explore the key molecular changes related to endoplasmic reticulum stress and mitochondrial damage in the uterine tissues of bitches with open pyometra.. A total of 16 bitches were included in the study, divided into a healthy control group (n = 8) and a pyometra group (n = 8).. The results showed that compared with healthy individuals, the mRNA expression. and protein abundance of endoplasmic reticulum stress marker genes PERK, ATF4, CHOP, GRP78 and GRP94 were significantly upregulated in the uterine tissues of bitches with pyometra.

Article Title: Biomimetic cancer cell membrane-coated liposomal nanocarriers loaded with silibinin suppress gastric cancer progression via SNHG1/miR-383-5p/HSP90AA1 axis-mediated PI3K/AKT pathway inhibition
Article Snippet: Equal amounts of protein (20–40 μg) were separated by 10 % SDS-PAGE and transferred to PVDF membranes (Cat. No. IPVH00010, Millipore, USA). .. Membranes were blocked at ambient temperature for 2 h with 5 % non-fat milk in PBS containing 0.1 % Tween-20 (PBST), followed by overnight incubation at 4 °C with the following primary antibodies: Rabbit anti-Pan-cadherin (1:1000, ab51034, Abcam, UK), Rabbit anti-COXIV (1:1000, 4850, Cell Signaling Technology, USA), Rabbit anti-Histone H3 (1:2000, 4499, CST, USA), Rabbit anti-Na + /K + -ATPase (1:1000, 3010, CST, USA), Rabbit anti-HSP90AA1 (1:500, ab314005, Abcam, UK), Rabbit anti-E-cadherin (1:1000, 3195S, CST, USA), Rabbit anti-PI3K (1:500, ab154598, Abcam, UK), Rabbit anti-p-PI3K (1:1000, ab191606, Abcam, UK), Rabbit anti-AKT (1:500, ab8805, Abcam, UK), Rabbit anti-p-AKT (1:500, ab38449, Abcam, UK), Rabbit anti-GAPDH (1:10000, 10494-1-AP, Proteintech, China; used as loading control). .. After washing with PBST, membranes were incubated with HRP-conjugated secondary antibody (1:5000, ab205718, Abcam, UK) for 2 h at room temperature.

other:

Article Title: CRP Deficiency Rescues Periodontitis-Induced Hippocampal Neurogenesis Impairment by Suppressing OPC-Derived BMP4 Signaling in Rats.
Article Snippet: Hippocampal tissues, gingival tissues, and primary cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (Epizyme, PC104) supplemented with protease and phosphatase inhibitors (Epizyme, GRF101 and GRF102; Beyotime, ST506) for protein extraction.

Control:

Image Search Results

3-HPA inhibits the secretion of inflammatory factors and glycolysis in macrophages (A and B) Schematic diagram of THP-1 cell (A) and BMDMs (B) activation into pro-inflammatory macrophages. Created with BioRender.com. (C) The concentrations of IL-6, TNF-α, and IL-1β in THP-1 cell supernatants were quantified by ELISA after 24 h treatment with LPS (100 ng/mL), or LPS+3-HPA (5 mM). Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test with ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. (D) The concentrations of IL-6, TNF-α, and IL-1β in supernatants of THP-1 cells treated with different concentrations of 3-HPA (0, 0.625, 1.25, 5 mM) followed by LPS stimulation. Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. (E) The concentrations of IL-6, TNF-α, and IL-1β in BMDM cell supernatants were quantified by ELISA after 24 h treatment with LPS (100 ng/mL) or LPS+3-HPA (5 mM). Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test with ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. (F) Bubble plot of KEGG pathway enrichment analysis for differentially expressed genes between LPS (100 ng/mL) and LPS+3-HPA (5 mM) treated in THP-1 cells. The size of each bubble represents the number of differentially expressed genes, and the color indicates the enrichment factor. (G and H) Pyruvate and lactate levels in THP-1 cells (G) and BMDMs (H) treated with LPS (100 ng/mL), or LPS+3-HPA (5 mM). (I) Immunoblots of protein expression levels of HK, GAPDH, PKM, and LDHA in THP-1 cells treated with LPS (100 ng/mL), or LPS+3-HPA (5 mM), and quantitative results of GAPDH. (J) The mRNA levels of GAPDH in THP-1 cells treated with LPS (100 ng/mL) or LPS+3-HPA (5 mM). (K) GAPDH activity assay in THP-1 cells and BMDMs treated with PBS, LPS (100 ng/mL), or LPS+3-HPA (5 mM). Data in (G–K) are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test with ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001; ns, not significant.

Journal: iScience

Article Title: 3-Hydroxypropionic acid converts inflammatory macrophage glycolysis into mitochondrial oxidation through GAPDH carboxyethylation

doi: 10.1016/j.isci.2026.116258

Figure Lengend Snippet: 3-HPA inhibits the secretion of inflammatory factors and glycolysis in macrophages (A and B) Schematic diagram of THP-1 cell (A) and BMDMs (B) activation into pro-inflammatory macrophages. Created with BioRender.com. (C) The concentrations of IL-6, TNF-α, and IL-1β in THP-1 cell supernatants were quantified by ELISA after 24 h treatment with LPS (100 ng/mL), or LPS+3-HPA (5 mM). Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test with ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. (D) The concentrations of IL-6, TNF-α, and IL-1β in supernatants of THP-1 cells treated with different concentrations of 3-HPA (0, 0.625, 1.25, 5 mM) followed by LPS stimulation. Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. (E) The concentrations of IL-6, TNF-α, and IL-1β in BMDM cell supernatants were quantified by ELISA after 24 h treatment with LPS (100 ng/mL) or LPS+3-HPA (5 mM). Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test with ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. (F) Bubble plot of KEGG pathway enrichment analysis for differentially expressed genes between LPS (100 ng/mL) and LPS+3-HPA (5 mM) treated in THP-1 cells. The size of each bubble represents the number of differentially expressed genes, and the color indicates the enrichment factor. (G and H) Pyruvate and lactate levels in THP-1 cells (G) and BMDMs (H) treated with LPS (100 ng/mL), or LPS+3-HPA (5 mM). (I) Immunoblots of protein expression levels of HK, GAPDH, PKM, and LDHA in THP-1 cells treated with LPS (100 ng/mL), or LPS+3-HPA (5 mM), and quantitative results of GAPDH. (J) The mRNA levels of GAPDH in THP-1 cells treated with LPS (100 ng/mL) or LPS+3-HPA (5 mM). (K) GAPDH activity assay in THP-1 cells and BMDMs treated with PBS, LPS (100 ng/mL), or LPS+3-HPA (5 mM). Data in (G–K) are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test with ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001; ns, not significant.

Article Snippet: Subsequently, cells were incubated overnight at 4°C with primary antibodies: rabbit anti-GAPDH polyclonal antibody (R1210-1, HUABIO, 1:200 dilution) and mouse anti-ceC247 monoclonal antibody (1:200 dilution).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Activity Assay

GAPDH is modified with carboxyethylation at Cys 247 (A) Mass spectrometry analysis of GAPDH peptide (234–260) with carboxyethylation and 3-HPA (5 mM) incubated with the GAPDH peptide (234–260) at 37 °C for 4 h. (B) SPR analysis of the affinity of the anti-ceC247 antibody for the carboxyethylation modified GAPDH peptide (234–260) and unmodified GAPDH peptide (234–260). (C) ELISA-based binding curve of anti-ceC247 antibody to modified GAPDH peptide (GAPDH ce (234–260)) and unmodified GAPDH peptide (234–260). Data are the means ± SD and n = 3 per group. Statistical significance was determined using two-way ANOVA followed by ∗∗p < 0.01. (D) Chemical structures of cysteine carboxyethylation and cysteine lactylation. (E) Unmodified GAPDH peptide (234–260), carboxyethylated peptide (GAPDH ce (234–260)), and lactylated peptide (GAPDH lac (234–260)) were tested with the anti-ceC247 antibody in dot blot assays. (F) Immunoblots of lysates from 293 T cells overexpressing GAPDH, which were treated with 5 mM 3-HPA and 5 μM MG132. The blots were probed with the anti-ceC247 antibody. (G) 3-HPA incubated with the GAPDH peptide (234–260) at 37 °C for 4 h. An anti-ceC247 antibody and the anti-wtC247 antibody were used in dot blot assays.

Journal: iScience

Article Title: 3-Hydroxypropionic acid converts inflammatory macrophage glycolysis into mitochondrial oxidation through GAPDH carboxyethylation

doi: 10.1016/j.isci.2026.116258

Figure Lengend Snippet: GAPDH is modified with carboxyethylation at Cys 247 (A) Mass spectrometry analysis of GAPDH peptide (234–260) with carboxyethylation and 3-HPA (5 mM) incubated with the GAPDH peptide (234–260) at 37 °C for 4 h. (B) SPR analysis of the affinity of the anti-ceC247 antibody for the carboxyethylation modified GAPDH peptide (234–260) and unmodified GAPDH peptide (234–260). (C) ELISA-based binding curve of anti-ceC247 antibody to modified GAPDH peptide (GAPDH ce (234–260)) and unmodified GAPDH peptide (234–260). Data are the means ± SD and n = 3 per group. Statistical significance was determined using two-way ANOVA followed by ∗∗p < 0.01. (D) Chemical structures of cysteine carboxyethylation and cysteine lactylation. (E) Unmodified GAPDH peptide (234–260), carboxyethylated peptide (GAPDH ce (234–260)), and lactylated peptide (GAPDH lac (234–260)) were tested with the anti-ceC247 antibody in dot blot assays. (F) Immunoblots of lysates from 293 T cells overexpressing GAPDH, which were treated with 5 mM 3-HPA and 5 μM MG132. The blots were probed with the anti-ceC247 antibody. (G) 3-HPA incubated with the GAPDH peptide (234–260) at 37 °C for 4 h. An anti-ceC247 antibody and the anti-wtC247 antibody were used in dot blot assays.

Techniques: Modification, Mass Spectrometry, Incubation, Enzyme-linked Immunosorbent Assay, Binding Assay, Dot Blot, Western Blot

3-HPA-induced carboxyethylation of GAPDH promotes its degradation through the ubiquitin-proteasome pathway (A) Chemical structure of carboxyethylated cysteine (left) and structures of aspartic acid (D), glutamic acid (E), methionine (M), and cysteine (C). (B) Immunoblot of GAPDH after transient transfection of flag-tagged GAPDH(C), GAPDH(D), GAPDH(M), GAPDH(E) plasmid in 293 T cells at 24 h, 36 h, and 48 h. (C) Immunoblot of GAPDH after CHX treatment. The GAPDH antibody was used to compare the degradation rates of GAPDH(M), GAPDH(E), GAPDH(D), and GAPDH(C). Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test ∗p < 0.05; ns, not significant. (D) Immunoblot and quantitative analysis of GAPDH ce after CHX treatment. The anti-ceC247 antibody and anti-GAPDH antibody were used to compare the degradation rates of carboxyethylated GAPDH. Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test ∗∗p < 0.01; ns, not significant. (E) Immunoblot and quantitative analysis of GAPDH ce after 3-HPA treatment. The anti-ceC247 antibody and anti-GAPDH antibody were used to compare the content of carboxyethylated GAPDH and total GAPDH. Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test ∗p < 0.05; ∗∗p < 0.01; ns, not significant. (F) Immunoblot and quantitative analysis of GAPDH ce in 293 T cells treated with 3-HPA (5 mM) combined with proteasomal inhibitor MG132, autophagic inhibitor Chloroquine, or lysosomal inhibitor Bafilomycin A1. Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test ∗∗p < 0.01; ∗∗∗∗p < 0.0001; ns, not significant. (G) Immunoprecipitation of GAPDH or GAPDH ce followed by immunoblotting for Myc in 293 T cells transfected with Myc-Ub. Cells were treated with 3-HPA (5 mM) and MG132 (5 μM) for 24 h. (H) Immunoprecipitation of GAPDH or GAPDH ce followed by immunoblotting for Myc in 293 T cells transfected with Myc-Ub mutants (K6O, K11O, K27O, K29O, K33O, K48O, K63O). Cells were treated with 3-HPA (5 mM) for 24 h.

Journal: iScience

Article Title: 3-Hydroxypropionic acid converts inflammatory macrophage glycolysis into mitochondrial oxidation through GAPDH carboxyethylation

doi: 10.1016/j.isci.2026.116258

Figure Lengend Snippet: 3-HPA-induced carboxyethylation of GAPDH promotes its degradation through the ubiquitin-proteasome pathway (A) Chemical structure of carboxyethylated cysteine (left) and structures of aspartic acid (D), glutamic acid (E), methionine (M), and cysteine (C). (B) Immunoblot of GAPDH after transient transfection of flag-tagged GAPDH(C), GAPDH(D), GAPDH(M), GAPDH(E) plasmid in 293 T cells at 24 h, 36 h, and 48 h. (C) Immunoblot of GAPDH after CHX treatment. The GAPDH antibody was used to compare the degradation rates of GAPDH(M), GAPDH(E), GAPDH(D), and GAPDH(C). Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test ∗p < 0.05; ns, not significant. (D) Immunoblot and quantitative analysis of GAPDH ce after CHX treatment. The anti-ceC247 antibody and anti-GAPDH antibody were used to compare the degradation rates of carboxyethylated GAPDH. Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test ∗∗p < 0.01; ns, not significant. (E) Immunoblot and quantitative analysis of GAPDH ce after 3-HPA treatment. The anti-ceC247 antibody and anti-GAPDH antibody were used to compare the content of carboxyethylated GAPDH and total GAPDH. Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test ∗p < 0.05; ∗∗p < 0.01; ns, not significant. (F) Immunoblot and quantitative analysis of GAPDH ce in 293 T cells treated with 3-HPA (5 mM) combined with proteasomal inhibitor MG132, autophagic inhibitor Chloroquine, or lysosomal inhibitor Bafilomycin A1. Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test ∗∗p < 0.01; ∗∗∗∗p < 0.0001; ns, not significant. (G) Immunoprecipitation of GAPDH or GAPDH ce followed by immunoblotting for Myc in 293 T cells transfected with Myc-Ub. Cells were treated with 3-HPA (5 mM) and MG132 (5 μM) for 24 h. (H) Immunoprecipitation of GAPDH or GAPDH ce followed by immunoblotting for Myc in 293 T cells transfected with Myc-Ub mutants (K6O, K11O, K27O, K29O, K33O, K48O, K63O). Cells were treated with 3-HPA (5 mM) for 24 h.

Techniques: Ubiquitin Proteomics, Western Blot, Transfection, Plasmid Preparation, Immunoprecipitation

3-HPA enhances the metabolite content of the TCA cycle and mitochondrial oxidation (A) Schematic diagram of THP-1 with 2-DG treatment. Created with BioRender.com. (B) Concentrations of IL-6, TNF-α, and IL-1β in supernatants of THP-1 cells treated with LPS, LPS+3-HPA, LPS+2-DG, or LPS+3-HPA+2-DG. Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test with ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001; ns, not significant. (C) Schematic diagram of THP-1 with high glucose treatment. Created with BioRender.com. (D) Concentrations of IL-6, TNF-α, and IL-1β in supernatants of THP-1 cells treated with LPS+3-HPA, LPS+3-HPA+glucose. Data are the means ± SD and n = 3 per group. Statistical significance was determined using unpaired Student’s t test with ∗∗∗p < 0.001; ns, not significant. (E) KEGG pathway enrichment analysis of metabolic pathways in BMDM cells treated with LPS or LPS+3-HPA. (F) Relative abundance of metabolite (ornithine, citrulline, L-malate, succinic acid, trans-aconitic acid, cis-aconitic acid) in BMDM cells treated with LPS or LPS+3-HPA. Data are the means ± SD and n = 4 per group. Statistical significance was determined using unpaired Student’s t test with ∗p < 0.05; ∗∗p < 0.01. (G) Correlation network of metabolites and genes in the metabolic pathway. Nodes represent metabolites (blue squares) and genes (colored circles). Gray edges indicate pairwise correlations between metabolites and genes. The colors similarly represent expression levels, with red typically indicating higher expression and blue indicating lower expression compared to the mean. (H) Schematic diagram of arginine metabolism and the TCA cycle. (I) Heatmap of mitochondrial oxidation-related gene expression associated with differentially expressed metabolites. Red indicates relatively high gene expression, while blue indicates relatively low gene expression within each row. (J) Schematic diagram of the catalytic function of the GAPDH enzyme. (K) Concentrations of the NAD + /NADH ratio in THP-1 cells and BMDM cells treated with PBS, LPS, or LPS+3-HPA. Data are the means ± SD and n = 4 per group. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test with ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗∗ p < 0.0001; (L) ATP concentrations in THP-1 cells and BMDMs treated with PBS, LPS, or LPS+3-HPA. Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test with ∗ p < 0.05; ∗∗∗∗ p < 0.0001; ns, not significant. (M) Representative images and mitochondrial analysis of BMDM cells treated with PBS, LPS, or LPS+3-HPA. Scale bars, 1 μm (upper) and 0.25 μm (lower). For mitochondrial number, n = 8–12 ( n = 12 for PBS, n = 8 for LPS, n = 9 for LPS+3-HPA group).

Journal: iScience

Article Title: 3-Hydroxypropionic acid converts inflammatory macrophage glycolysis into mitochondrial oxidation through GAPDH carboxyethylation

doi: 10.1016/j.isci.2026.116258

Figure Lengend Snippet: 3-HPA enhances the metabolite content of the TCA cycle and mitochondrial oxidation (A) Schematic diagram of THP-1 with 2-DG treatment. Created with BioRender.com. (B) Concentrations of IL-6, TNF-α, and IL-1β in supernatants of THP-1 cells treated with LPS, LPS+3-HPA, LPS+2-DG, or LPS+3-HPA+2-DG. Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test with ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001; ns, not significant. (C) Schematic diagram of THP-1 with high glucose treatment. Created with BioRender.com. (D) Concentrations of IL-6, TNF-α, and IL-1β in supernatants of THP-1 cells treated with LPS+3-HPA, LPS+3-HPA+glucose. Data are the means ± SD and n = 3 per group. Statistical significance was determined using unpaired Student’s t test with ∗∗∗p < 0.001; ns, not significant. (E) KEGG pathway enrichment analysis of metabolic pathways in BMDM cells treated with LPS or LPS+3-HPA. (F) Relative abundance of metabolite (ornithine, citrulline, L-malate, succinic acid, trans-aconitic acid, cis-aconitic acid) in BMDM cells treated with LPS or LPS+3-HPA. Data are the means ± SD and n = 4 per group. Statistical significance was determined using unpaired Student’s t test with ∗p < 0.05; ∗∗p < 0.01. (G) Correlation network of metabolites and genes in the metabolic pathway. Nodes represent metabolites (blue squares) and genes (colored circles). Gray edges indicate pairwise correlations between metabolites and genes. The colors similarly represent expression levels, with red typically indicating higher expression and blue indicating lower expression compared to the mean. (H) Schematic diagram of arginine metabolism and the TCA cycle. (I) Heatmap of mitochondrial oxidation-related gene expression associated with differentially expressed metabolites. Red indicates relatively high gene expression, while blue indicates relatively low gene expression within each row. (J) Schematic diagram of the catalytic function of the GAPDH enzyme. (K) Concentrations of the NAD + /NADH ratio in THP-1 cells and BMDM cells treated with PBS, LPS, or LPS+3-HPA. Data are the means ± SD and n = 4 per group. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test with ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗∗ p < 0.0001; (L) ATP concentrations in THP-1 cells and BMDMs treated with PBS, LPS, or LPS+3-HPA. Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test with ∗ p < 0.05; ∗∗∗∗ p < 0.0001; ns, not significant. (M) Representative images and mitochondrial analysis of BMDM cells treated with PBS, LPS, or LPS+3-HPA. Scale bars, 1 μm (upper) and 0.25 μm (lower). For mitochondrial number, n = 8–12 ( n = 12 for PBS, n = 8 for LPS, n = 9 for LPS+3-HPA group).

Techniques: Expressing, Gene Expression

GAPDH carboxyethylation inhibited macrophage glycolysis and the release of inflammatory factors (A) Schematic workflow illustrating the strategy of silencing endogenous GAPDH via 3′UTR-targeting siRNA and overexpressing exogenous GAPDH. (B) Immunoblot and quantitative analysis of GAPDH protein in 293 T cells transfected with GAPDH 3′UTR-targeting siRNAs (siGAPDH 1, siGAPDH 2) or siRNA NC. Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test ∗∗p < 0.01. (C) Relative mRNA expression of GAPDH in 293 T cells transfected with GAPDH 3′UTR-targeting siRNAs (siGAPDH 1, siGAPDH 2) or siRNA NC. Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test ∗∗∗∗p < 0.0001. (D) Immunoblot analysis of FLAG-tagged exogenous GAPDH(E) and GAPDH in 293 T cells. Knockdown of endogenous GAPDH with siRNA followed by the overexpression of GAPDH (E). Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test with ∗p < 0.05; ns, not significant. (E) Relative mRNA expression of GAPDH in 293 T cells knockdowned endogenous GAPDH (siGAPDH) and overexpressed GAPDH (C) and GAPDH (E), respectively. Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test with ∗∗p < 0.01; ∗∗∗p < 0.001; ns, not significant. (F) GAPDH activity assay in 293 T cells transfected with GAPDH 3′UTR siRNA, and overexpressing GAPDH(C), GAPDH(E). Data are the means ± SD and n = 4 per group. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test with ∗p < 0.05; ∗∗∗∗p < 0.0001; ns, not significant. (G) Concentrations of lactate and pyruvate in 293 T cells transfected with GAPDH 3′UTR siRNA, and overexpressing GAPDH(C), GAPDH(E). Data are the means ± SD and n = 4 per group. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test with ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗∗p < 0.0001. (H) Relative mRNA expression of IL-6 , TNF-α , and IL-1β in THP-1 cells which transfected with GAPDH 3′UTR siRNA, and overexpressing GAPDH(C) or GAPDH(E). Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test with ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ns, not significant. (I) The concentration of TNF-α in THP-1 cells that overexpressed GAPDH(C) or GAPDH(E). Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test with ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001; ns, not significant.

Journal: iScience

Article Title: 3-Hydroxypropionic acid converts inflammatory macrophage glycolysis into mitochondrial oxidation through GAPDH carboxyethylation

doi: 10.1016/j.isci.2026.116258

Figure Lengend Snippet: GAPDH carboxyethylation inhibited macrophage glycolysis and the release of inflammatory factors (A) Schematic workflow illustrating the strategy of silencing endogenous GAPDH via 3′UTR-targeting siRNA and overexpressing exogenous GAPDH. (B) Immunoblot and quantitative analysis of GAPDH protein in 293 T cells transfected with GAPDH 3′UTR-targeting siRNAs (siGAPDH 1, siGAPDH 2) or siRNA NC. Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test ∗∗p < 0.01. (C) Relative mRNA expression of GAPDH in 293 T cells transfected with GAPDH 3′UTR-targeting siRNAs (siGAPDH 1, siGAPDH 2) or siRNA NC. Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test ∗∗∗∗p < 0.0001. (D) Immunoblot analysis of FLAG-tagged exogenous GAPDH(E) and GAPDH in 293 T cells. Knockdown of endogenous GAPDH with siRNA followed by the overexpression of GAPDH (E). Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test with ∗p < 0.05; ns, not significant. (E) Relative mRNA expression of GAPDH in 293 T cells knockdowned endogenous GAPDH (siGAPDH) and overexpressed GAPDH (C) and GAPDH (E), respectively. Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test with ∗∗p < 0.01; ∗∗∗p < 0.001; ns, not significant. (F) GAPDH activity assay in 293 T cells transfected with GAPDH 3′UTR siRNA, and overexpressing GAPDH(C), GAPDH(E). Data are the means ± SD and n = 4 per group. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test with ∗p < 0.05; ∗∗∗∗p < 0.0001; ns, not significant. (G) Concentrations of lactate and pyruvate in 293 T cells transfected with GAPDH 3′UTR siRNA, and overexpressing GAPDH(C), GAPDH(E). Data are the means ± SD and n = 4 per group. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test with ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗∗p < 0.0001. (H) Relative mRNA expression of IL-6 , TNF-α , and IL-1β in THP-1 cells which transfected with GAPDH 3′UTR siRNA, and overexpressing GAPDH(C) or GAPDH(E). Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test with ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ns, not significant. (I) The concentration of TNF-α in THP-1 cells that overexpressed GAPDH(C) or GAPDH(E). Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test with ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001; ns, not significant.

Techniques: Western Blot, Transfection, Expressing, Knockdown, Over Expression, Activity Assay, Concentration Assay

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