mouse monoclonal anti dnmt1 antibody  (Novus Biologicals)

Journal: eLife

Article Title: Maternal obesity may disrupt offspring metabolism by inducing oocyte genome hyper-methylation via increased DNMTs

doi: 10.7554/eLife.97507

Figure Lengend Snippet: ( A ) The expression levels of Dnmt1 , Dnmt3a, and Dnmt3l in oocytes were examined using qPCR after the treatment with SQ22536 and forskolin. *p<0.05. Data presented as mean ± SEM; a two-tail t-test was used. p-value presented in the source data. ( B ) The relative expressions of Dnmt1 , Dnmt3a, and Dnmt3l in oocytes were examined using qPCR after the treatment with luzindole and melatonin. *p<0.05. Data presented as mean ± SEM; a two-tail t-test was used. ( C ) After 8-Bromo-cAMP treatment, the relative expression of DNMT3a in oocytes was examined using immunofluorescence and calculated by Image J ( D ) (Control, n=54; 8-Bromo-cAMP, n=70, p=0.002447). **p<0.01. Data presented as mean ± SEM; a two-tail t-test was used. ( E and F ) Treatment with the protein kinase A (PKA) antagonist H 89 2HCL treatment significantly reduced the level of DNMT3A in oocytes examined using immunofluorescence (Control, n=62; H 89 2HCl, n=48, p=0.003922). **p<0.01. Data presented as mean ± SEM; a two-tail t-test was used. ( G and H ) DNMT1 localization in the oocyte nucleus was examined using immunofluorescence after 8-Bromo-cAMP treatment (Control, n=30; 8-Bromo-cAMP, n=31, p=3.136*10 –7 ). ***p<0.001. Data presented as mean ± SEM;a two-tail t-test was used. ( I and J ) The localization of DNMT1 in oocyte nucleus was reduced by the treatment with the PKA antagonist H 89 2HCL (Control, n=22; H 89 2HCl, n=28, p=0.004929). ** p<0.01. Data presented as mean ± SEM; a two-tail t-test was used. Source data are presented in . Figure 7—source data 1. Extended data for .

Article Snippet: Antibody , Anti-DNMT1 antibody (Mouse Monoclonal) , Active motif , 39204 , IF(1:200).

Techniques: Expressing, Immunofluorescence, Control

Journal: eLife

Article Title: Maternal obesity may disrupt offspring metabolism by inducing oocyte genome hyper-methylation via increased DNMTs

doi: 10.7554/eLife.97507

Figure Lengend Snippet: ( A ) The relative expression of Dnmt1 , Dnmt3a, and Dnmt3l in HFD oocytes was examined using qPCR. *p<0.05; **p<0.01. Data presented as mean ± SD; two-tail t-test used. p-value presented in the source data. ( B ) The concentration of cAMP in HFD oocytes was examined using ELISA. **p<0.01. Data presented as mean ± SEM; a two-tail t-test was used, n: CD=6, HFD = 3, p=0.004375.( C ) The relative expressions CREB1 and CREM in HFD oocytes were tested using qPCR. *p<0.05; **p<0.01. Data presented as mean ± SD; a two-tail t-test was used. Replicated three times for each gene, and p value presented in the source data.( D and E ) The level of pCREB1 in oocytes was examined using immunofluorescence, and the relative fluorescence intensity was calculated by Image J ( E ) (CD, n=69; HFD, n=49, p=3.326×10 –16 ; HFD + melatonin, n=61, p=8.997×10 –20 ). HFD, oocytes from obese mice; CD, oocytes from control mice; HFD + melatonin, oocytes from obese mice treated with exogenous melatonin. *p<0.05; ***p<0.001. Data presented as mean ± SEM; a two-tail t-test was used. ( F and G ) Treatment with the PKA antagonist H89 2HCL reduced the methylation level of HFD oocytes (CD, n=48; HFD, n=31, p=1.674*10 –13 ; HFD + H 89 2HCl, n=27, p=0.00324). ** p<0.01; *** p<0.001. Data presented as mean ± SEM; a two-tail t-test was used. ( H and I ) The level of pCREB1 in HFD oocytes was also decreased by the treatment with the protein kinase A (PKA) antagonist H89 2HCL (CD, n=17; HFD, n=17, p=0.006249; HFD +H 89 2HCl, n=22, p=0.027987). *p<0.05; **p<0.01; ns, no statistical significance between groups. Data presented as mean ± SEM; a two-tail t-test was used. ( J and K ) Treatment with the PKA antagonist H89 2HCL reduced the localization of DNMT1 in HFD oocytes (CD, n=24; HFD, n=29, p=6.214×10 –6 ; HFD + H 89 2HCl, n=25, p0.003147). **p<0.01; ***p<0.001; ns, no statistical significance between groups. Data presented as mean ± SEM; a two-tail t-test was used. Source data are presented in . Figure 8—source data 1. Extended data for .

Article Snippet: Antibody , Anti-DNMT1 antibody (Mouse Monoclonal) , Active motif , 39204 , IF(1:200).

Techniques: Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Fluorescence, Control, Methylation

Journal: eLife

Article Title: Maternal obesity may disrupt offspring metabolism by inducing oocyte genome hyper-methylation via increased DNMTs

doi: 10.7554/eLife.97507

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-DNMT1 antibody (Mouse Monoclonal) , Active motif , 39204 , IF(1:200).

Techniques: Sequencing, Enzyme-linked Immunosorbent Assay, Software

Journal: Frontiers in Immunology

Article Title: Amelioration effect of 18β-Glycyrrhetinic acid on methylation inhibitors in hepatocarcinogenesis -induced by diethylnitrosamine

doi: 10.3389/fimmu.2023.1206990

Figure Lengend Snippet: Measurement of the cofactor of methylation activity (DNMT1and MS) by qRT-PCR and flow cytometery. Positive control increased DNMT1 to 7-fold change while other treatment makes significant downregulation (A) . Error bars indicate standard deviation of three independent experiments. The student’s two-tailed t-test was used for statistical analysis. P-value ≤ 0.05 was considered statistically significant. Positive control increased MS to 10-fold change while other treatment makes significant downregulation (B) . Protein expression profile of DNMT1 and MS indicated by red and blue dotes using secondary antibodies Alex Flour-488 and 594, respectively (C) . *statistically significant difference as compared with the controls (P, 0.05 for each). While, ** statistically significant difference as compared with the controls (P, 0.01 for each).

Article Snippet: The same procedures were considered for staining DNMT1, MS, NFkB, and STAT3 using specific antibodies: mouse monoclonal anti-DNMT1 (Sigma-Aldrich, Germany), rabbit monoclonal anti-MS (Sigma-Aldrich, Germany), mouse monoclonal anti-NFkB (Sigma-Aldrich, Germany), and rabbit monoclonal anti-STAT3 (Sigma-Aldrich, Germany), respectively.

Techniques: Methylation, Activity Assay, Quantitative RT-PCR, Positive Control, Standard Deviation, Two Tailed Test, Expressing

Journal: iScience

Article Title: Prothymosin α accelerates dengue virus-induced thrombocytopenia

doi: 10.1016/j.isci.2023.108422

Figure Lengend Snippet: DENV infection reduces miR-126-3p, increases DNMT1, and reduces GATA-1 expression in Meg-01 cells (A and B) Detection and quantification of miR-126-3p (A) as well as DNMT1, viral NS3, and GATA-1 proteins in Meg-01 cells infected with DENV-2 at an MOI of 1 at different time points by RT-qPCR (A) and immunoblotting (B). Values are means ± SD (n = 4, one-way ANOVA). The ratio of mock infected cells was arbitrarily set to 100 (A). Expression of GAPDH served as the loading control (B). Values shown below the blots are ratios between the intensity of the bands corresponding to DNMT1 or GATA-1 and those corresponding to GAPDH analyzed by densitometry, where the ratio of mock infected cells was set to 1. (C) Immunohistochemical detection (original magnification ×200; scale bar = 100 μm) (left) and quantitation of DNMT1-positive cells (right) in the bone marrow of WT mice treated with either saline or DENV-2 + anti-CD41 at day 4 pi. Values shown are mean ± SD (n = 3, Student’s t test). (D–G) Detection and quantification of miR-126-3p (D), DNMT1 (E), GATA-1 (F), and ProT (G) in Meg-01 cells transduced with LV.miR-126 or LV.miR-Ctrl by RT-qPCR. The ratio of LV.miR-Ctrl-transduced control cells was arbitrarily set to 100. Values shown are mean ± SD (n = 4, Student’s t test).

Article Snippet: Mouse monoclonal anti-Dnmt1 antibody , Santa Cruz Biotechnology , Cat #sc-271729; RRID: AB_10710384.

Techniques: Infection, Expressing, Quantitative RT-PCR, Western Blot, Control, Immunohistochemical staining, Quantitation Assay, Saline, Transduction

Journal: iScience

Article Title: Prothymosin α accelerates dengue virus-induced thrombocytopenia

doi: 10.1016/j.isci.2023.108422

Figure Lengend Snippet: DNMT1 negatively regulates GATA-1 expression, and GATA-1 also negatively regulates ProT expression (A) Detection (left) and quantification (right) of GATA-1 and viral NS3 in DENV-infected Meg-01 cells treated with 1, 2, or 5 μM of the DNMT1 inhibitor 5-AZA-2′-deoxycytidine (5′-Aza-dc) for 72 h. Expression of β-actin served as the loading control. The immunoblot is from one representative experiment of three (left). Values shown are ratios between the intensity of the bands corresponding to GATA-1 and those corresponding to β-actin analyzed by densitometry, where the ratio of mock infected cells was set to 1 (right). Values shown are mean ± SD (n = 3, one-way ANOVA). (B) ChIP assay showing a 2.5-fold increase in the binding of DNMT1 to the GATA-1 promoter region in Meg-01 cells after infection with DENV-2 at an MOI of 1 for 3 days. Cross-linked chromatin was immunoprecipitated with anti-DNMT-1 or anti-IgG antibody combined with protein G agarose beads, followed by PCR amplification of the GATA-1 promoter. The ratio shown below the images is normalized to the amount of the input. (C) The PTMA promoter activity assessed in 293T cells 48 h after cotransfection with 1 μg of pGL3-pProT-Luc and various doses of pSin4-EF1a-GATA-1-IRES-Puro (upper panel). The total amount of plasmid DNA for transfection was kept constant by the addition of a control plasmid. Validation of GATA-1 expression in a dose-dependent manner by immunoblotting (lower panel). Expression of β-actin served as the loading control. Values shown are means ± SD (n = 3, one-way ANOVA). (D) Expression of ProT (upper panel) and validation of knockdown of GATA-1 expression (lower panel) in GATA-1 knockdown Meg-01 and control cells. Meg-01 cells were transduced with lentiviral vectors encoding shRNAs specific to GATA-1 (shGATA-1-358 and shGATA-1-359) or LacZ (control). Expression of β-actin or GAPDH served as the loading control. Values shown below the blots are ratios between the intensity of the bands corresponding to ProT or GATA-1 and those corresponding to β-actin or GAPDH analyzed by densitometry, where the ratio of mock infected cells was set to 1.

Article Snippet: Mouse monoclonal anti-Dnmt1 antibody , Santa Cruz Biotechnology , Cat #sc-271729; RRID: AB_10710384.

Techniques: Expressing, Infection, Control, Western Blot, Binding Assay, Immunoprecipitation, Amplification, Activity Assay, Cotransfection, Plasmid Preparation, Transfection, Biomarker Discovery, Knockdown, Transduction

Journal: iScience

Article Title: Prothymosin α accelerates dengue virus-induced thrombocytopenia

doi: 10.1016/j.isci.2023.108422

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-Dnmt1 antibody , Santa Cruz Biotechnology , Cat #sc-271729; RRID: AB_10710384.

Techniques: Recombinant, Stripping Membranes, Isolation, Reverse Transcription, TaqMan microRNA Assay, Plasmid Preparation, shRNA, Software, RNA Expression, Knock-Out, Microarray

Journal: iScience

Article Title: Prothymosin α accelerates dengue virus-induced thrombocytopenia

doi: 10.1016/j.isci.2023.108422

Figure Lengend Snippet: DENV infection reduces miR-126-3p, increases DNMT1, and reduces GATA-1 expression in Meg-01 cells (A and B) Detection and quantification of miR-126-3p (A) as well as DNMT1, viral NS3, and GATA-1 proteins in Meg-01 cells infected with DENV-2 at an MOI of 1 at different time points by RT-qPCR (A) and immunoblotting (B). Values are means ± SD (n = 4, one-way ANOVA). The ratio of mock infected cells was arbitrarily set to 100 (A). Expression of GAPDH served as the loading control (B). Values shown below the blots are ratios between the intensity of the bands corresponding to DNMT1 or GATA-1 and those corresponding to GAPDH analyzed by densitometry, where the ratio of mock infected cells was set to 1. (C) Immunohistochemical detection (original magnification ×200; scale bar = 100 μm) (left) and quantitation of DNMT1-positive cells (right) in the bone marrow of WT mice treated with either saline or DENV-2 + anti-CD41 at day 4 pi. Values shown are mean ± SD (n = 3, Student’s t test). (D–G) Detection and quantification of miR-126-3p (D), DNMT1 (E), GATA-1 (F), and ProT (G) in Meg-01 cells transduced with LV.miR-126 or LV.miR-Ctrl by RT-qPCR. The ratio of LV.miR-Ctrl-transduced control cells was arbitrarily set to 100. Values shown are mean ± SD (n = 4, Student’s t test).

Article Snippet: Mouse monoclonal anti-Dnmt1 antibody , Abcam , Cat #ab13537; RRID: AB_300438.

Techniques: Infection, Expressing, Quantitative RT-PCR, Western Blot, Immunohistochemistry, Quantitation Assay, Saline, Transduction

Journal: iScience

Article Title: Prothymosin α accelerates dengue virus-induced thrombocytopenia

doi: 10.1016/j.isci.2023.108422

Figure Lengend Snippet: DNMT1 negatively regulates GATA-1 expression, and GATA-1 also negatively regulates ProT expression (A) Detection (left) and quantification (right) of GATA-1 and viral NS3 in DENV-infected Meg-01 cells treated with 1, 2, or 5 μM of the DNMT1 inhibitor 5-AZA-2′-deoxycytidine (5′-Aza-dc) for 72 h. Expression of β-actin served as the loading control. The immunoblot is from one representative experiment of three (left). Values shown are ratios between the intensity of the bands corresponding to GATA-1 and those corresponding to β-actin analyzed by densitometry, where the ratio of mock infected cells was set to 1 (right). Values shown are mean ± SD (n = 3, one-way ANOVA). (B) ChIP assay showing a 2.5-fold increase in the binding of DNMT1 to the GATA-1 promoter region in Meg-01 cells after infection with DENV-2 at an MOI of 1 for 3 days. Cross-linked chromatin was immunoprecipitated with anti-DNMT-1 or anti-IgG antibody combined with protein G agarose beads, followed by PCR amplification of the GATA-1 promoter. The ratio shown below the images is normalized to the amount of the input. (C) The PTMA promoter activity assessed in 293T cells 48 h after cotransfection with 1 μg of pGL3-pProT-Luc and various doses of pSin4-EF1a-GATA-1-IRES-Puro (upper panel). The total amount of plasmid DNA for transfection was kept constant by the addition of a control plasmid. Validation of GATA-1 expression in a dose-dependent manner by immunoblotting (lower panel). Expression of β-actin served as the loading control. Values shown are means ± SD (n = 3, one-way ANOVA). (D) Expression of ProT (upper panel) and validation of knockdown of GATA-1 expression (lower panel) in GATA-1 knockdown Meg-01 and control cells. Meg-01 cells were transduced with lentiviral vectors encoding shRNAs specific to GATA-1 (shGATA-1-358 and shGATA-1-359) or LacZ (control). Expression of β-actin or GAPDH served as the loading control. Values shown below the blots are ratios between the intensity of the bands corresponding to ProT or GATA-1 and those corresponding to β-actin or GAPDH analyzed by densitometry, where the ratio of mock infected cells was set to 1.

Article Snippet: Mouse monoclonal anti-Dnmt1 antibody , Abcam , Cat #ab13537; RRID: AB_300438.

Techniques: Expressing, Infection, Western Blot, Binding Assay, Immunoprecipitation, Amplification, Activity Assay, Cotransfection, Plasmid Preparation, Transfection, Transduction

Journal: iScience

Article Title: Prothymosin α accelerates dengue virus-induced thrombocytopenia

doi: 10.1016/j.isci.2023.108422

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-Dnmt1 antibody , Abcam , Cat #ab13537; RRID: AB_300438.

Techniques: Recombinant, Stripping Membranes, Isolation, TaqMan microRNA Assay, Plasmid Preparation, shRNA, Software, RNA Expression, Knock-Out, Microarray

Journal: eLife

Article Title: Inhibition of DNMT1 methyltransferase activity via glucose-regulated O -GlcNAcylation alters the epigenome

doi: 10.7554/eLife.85595

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-DNMT1 (60B1220.1) (mouse monoclonal) , Novus Biologicals , Cat# NB100-56519 , IP (1:250) WB (1:1000).

Techniques: Transfection, Construct, Plasmid Preparation, Sequencing, Mutagenesis, Activity Assay, Enzyme-linked Immunosorbent Assay, DNA Methylation Assay, Software, Staining, Marker