rabbit polyclonal anti gapdh  (Proteintech)

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

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.

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

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.

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

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).

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: Expressing, Gene Expression

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.

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

Journal: Oncology Letters

Article Title: RNA methyltransferase NSUN2 enhances vasculogenic mimicry and malignant progression of cervical cancer through upregulation of MMP-9

doi: 10.3892/ol.2026.15518

Figure Lengend Snippet: MMP-9 , a factor that promotes Vasculogenic mimicry, is highly expressed in CC and is associated with poor prognosis. (A) CC database of TCGA was used to analyze key factors associated with VM. (B) Association of Sox2 expression with overall survival in CC (log-rank test). (C) Association of MMP-9 expression with overall survival in CC (log-rank test). (D) Panoramic scans after immunohistochemical detection of MMP-9 and H&E staining in samples from cancerous and paracancerous tissues from subjects with CC. Scale bar, 50 µm. Original magnification, ×20. (E) Protein levels of MMP-9 in 20 paired samples, with the MMP-9 level in CC tissue expressed compared with that in the paired normal tissue. (F) Expression levels of MMP-9 mRNA in 44 paired CC and paracancerous tissues, with MMP-9 expression in CC tissue expressed compared with that in the paired normal tissue. (G) Comparison of the average expression levels of MMP-9 mRNA in CC tissues compared with paracancerous tissues. (H) HeLa and SiHa cells were incubated under hypoxia (0.1% O 2 ) and proteins collected at 24, 48 and 72 h for western blotting of ALDH1, EPHA2, MMP-9 and GAPDH. ImageJ was used to semi-quantify western blotting signals from HeLa (I) and SiHa (J) cells. GAPDH served as an internal reference. *P<0.05, **P<0.01 and ***P<0.001. MMP-9, matrix metalloproteinase 9; VM, vasculogenic mimicry; ALDH1, aldehyde dehydrogenase 1; EPHA2, ephrin type-A receptor 2; TCGA, The Cancer Genome Atlas; Sox2, SRY-box transcription factor 2; CC, cervical cancer; CESC, cervical squamous cell carcinoma.

Article Snippet: The membranes were blocked with 5% milk at 20±5°C for 1 h. The blocked membranes were incubated at 4°C overnight with the following antibodies: A rabbit monoclonal anti-NSUN2 antibody (1:1,000; cat. no. AB259941; Abcam), a rabbit monoclonal anti-transfer RNA aspartic acid methyltransferase 1 (TRDMT1) antibody (1:1,000; cat. no. 19221-1-AP; Proteintech Group, Inc.; Wuhan Sanying Biotechnology), a rabbit polyclonal anti-MMP-9 antibody (1:1,000; cat. no. 10375-2-AP; Proteintech Group, Inc.; Wuhan Sanying Biotechnology), a rabbit polyclonal anti-aldehyde dehydrogenase 1 (ALDH1) antibody (1:1,000; cat. no. 15910-1-AP; Proteintech Group, Inc.; Wuhan Sanying Biotechnology), a rabbit polyclonal anti-ephrin type-A receptor 2 (EPHA2) antibody (1:1,000; cat. no. AF5 238; Affinity Biosciences) and a rabbit polyclonal anti-GAPDH antibody (1:1,000; TA309157 OriGene Technologies, Inc.).

Techniques: Expressing, Immunohistochemical staining, Staining, Comparison, Incubation, Western Blot

Journal: Oncology Letters

Article Title: RNA methyltransferase NSUN2 enhances vasculogenic mimicry and malignant progression of cervical cancer through upregulation of MMP-9

doi: 10.3892/ol.2026.15518

Figure Lengend Snippet: m 5 C RNA methyltransferase NUSN2 is significantly upregulated in CC and its gene expression is associated with poor prognosis. (A) The level of m 5 C modification of RNA in CC cells was analyzed using a dot blot assay. Methylene blue staining served as an internal reference. (B) Semi-quantitative analysis of dot blot results in HeLa cells. (C) Semi-quantitative analysis of dot blot results in SiHa cells. (D) Expression levels of genes encoding m 5 C methyltransferases was analyzed within a CC database of TCGA. (E) HeLa and SiHa cells were incubated under hypoxia (0.1% O 2 ) and proteins collected at 24, 48 and 72 h were analyzed for NSUN2, TRDMT1 and GAPDH by western blotting. ImageJ was used to quantify western blotting signals from (F) HeLa and (G) SiHa cells. GAPDH served as an internal reference. (H) mRNA expression level of NSUN2 in 44 pairs of CC and paracancerous tissues were quantified using RT-qPCR. Gene expression in cancer tissues is expressed compared with expression in normal tissues. (I) Comparison of the average expression level of NSUN2 mRNA in CC tissues compared with paracancerous tissues. (J) Panoramic scans after IHC detection of NSUN2 and H&E staining in CC and paracancerous tissues. Scale bar, 50 µm. Magnification, ×20. (K) Protein levels of NSUN2 in 20 paired CC and paracancerous tissues, with the NSUN2 level in CC tissue expressed compared with that in the paired normal tissue. (L) Level of NSUN2 expression in CC was associated with a poor prognosis in the form of biochemical progression-free survival (P=0.018; log-rank test). *P<0.05, **P<0.01 and ***P<0.001. MMP9, matrix metalloproteinase 9; VM, vasculogenic mimicry; CC, cervical cancer; IHC, immunohistochemistry; NSUN2, NOP2/Sun RNA methyltransferase 2; m 5 C, 5-methylcytidine; TRDMT1, transfer RNA aspartic acid methyltransferase 1; TCGA, The Cancer Genome Atlas; RT-qPCR, reverse transcription-quantitative PCR; IHC, immunohistochemistry.

Article Snippet: The membranes were blocked with 5% milk at 20±5°C for 1 h. The blocked membranes were incubated at 4°C overnight with the following antibodies: A rabbit monoclonal anti-NSUN2 antibody (1:1,000; cat. no. AB259941; Abcam), a rabbit monoclonal anti-transfer RNA aspartic acid methyltransferase 1 (TRDMT1) antibody (1:1,000; cat. no. 19221-1-AP; Proteintech Group, Inc.; Wuhan Sanying Biotechnology), a rabbit polyclonal anti-MMP-9 antibody (1:1,000; cat. no. 10375-2-AP; Proteintech Group, Inc.; Wuhan Sanying Biotechnology), a rabbit polyclonal anti-aldehyde dehydrogenase 1 (ALDH1) antibody (1:1,000; cat. no. 15910-1-AP; Proteintech Group, Inc.; Wuhan Sanying Biotechnology), a rabbit polyclonal anti-ephrin type-A receptor 2 (EPHA2) antibody (1:1,000; cat. no. AF5 238; Affinity Biosciences) and a rabbit polyclonal anti-GAPDH antibody (1:1,000; TA309157 OriGene Technologies, Inc.).

Techniques: Gene Expression, Modification, Dot Blot, Staining, Quantitative Dot Blot, Expressing, Incubation, Western Blot, Quantitative RT-PCR, Comparison, Immunohistochemistry, Reverse Transcription, Real-time Polymerase Chain Reaction