primary antibodies against t- bet, gata3, dnmt1, βactin, foxo3, p- p65, p65 and laminb  (Proteintech)

Journal: Neurotherapeutics

Article Title: LINE-1 hypomethylation in cell-free DNA of high-grade glioma patients correlates with tissue levels and is associated with reduced DNMT1 and H4K20me3 expression

doi: 10.1016/j.neurot.2025.e00754

Figure Lengend Snippet: A Immunohistochemical expression of DNMT1, H4K20me3, H3K9me3 in normal brain tissues, grade 2 and grade 4 astrocytomas, respectively. Normal brain tissue displays lower DNMT1, H4K20me3 and H3K9me3 immunostaining than in astrocytic tumors. Within tumors, DNMT1, H4K20me3 and H3K9me3 immunostaining was reduced from grade 2 to grade 4. B DNMT1, H4K20me3, H3K9me3 H -score in normal brain and astrocytomas tissues in respect to histological grade. DNMT1, H4K20me3 and H3K9me3 expressions were significantly elevated in grade 2 in comparison to grade 4 astrocytomas. C Detection and quantification of DNMT1, H4K20me3 and H3K9me3 expression in grade 2 and 4 astrocytomas as well as normal brain tissues. All experiments were repeated at least three times and statistical analysis of the protein levels from three experiments (normalized to actin levels) is shown. ∗ p ​< ​0.05, ∗∗∗ p ​< ​0.001.

Article Snippet: Incubation of all the sections was performed overnight with monoclonal primary DNMT1 antibody (sc-271729, Santa Cruz Biotechnology, Dallas, USA, 1:100) or H4K20me3 (Ab 78517, Abcam, Cambridge, UK, 1:300) or H3K9me3 (CMA 308, Merck Millipore, MA, USA, 1:100).

Techniques: Immunohistochemical staining, Expressing, Immunostaining, Comparison

Journal: Neurotherapeutics

Article Title: LINE-1 hypomethylation in cell-free DNA of high-grade glioma patients correlates with tissue levels and is associated with reduced DNMT1 and H4K20me3 expression

doi: 10.1016/j.neurot.2025.e00754

Figure Lengend Snippet: Correlations among percentage of LINE-1 methylation and DNMT1 ( A ) and H4K20me3 ( B ) H -score in the entire cohort. Positive correlation of H4K20me3 H -score with DNMT1 ( C ) and H3K9me3 H -score ( D ) as well as H3K9me3 and DNMT1 ( E ) in the entire cohort.

Article Snippet: Incubation of all the sections was performed overnight with monoclonal primary DNMT1 antibody (sc-271729, Santa Cruz Biotechnology, Dallas, USA, 1:100) or H4K20me3 (Ab 78517, Abcam, Cambridge, UK, 1:300) or H3K9me3 (CMA 308, Merck Millipore, MA, USA, 1:100).

Techniques: Methylation

Journal: Neurotherapeutics

Article Title: LINE-1 hypomethylation in cell-free DNA of high-grade glioma patients correlates with tissue levels and is associated with reduced DNMT1 and H4K20me3 expression

doi: 10.1016/j.neurot.2025.e00754

Figure Lengend Snippet: Kaplan-Meier survival curves for LINE-1 meth ( A ), DNMT1 ( B ) and H4K20me3 ( C ), H3K9me3 ( D ) in the entire cohort (blue line ​= ​low expression, red line ​= ​high expression).

Article Snippet: Incubation of all the sections was performed overnight with monoclonal primary DNMT1 antibody (sc-271729, Santa Cruz Biotechnology, Dallas, USA, 1:100) or H4K20me3 (Ab 78517, Abcam, Cambridge, UK, 1:300) or H3K9me3 (CMA 308, Merck Millipore, MA, USA, 1:100).

Techniques: Expressing

Journal: Neurotherapeutics

Article Title: LINE-1 hypomethylation in cell-free DNA of high-grade glioma patients correlates with tissue levels and is associated with reduced DNMT1 and H4K20me3 expression

doi: 10.1016/j.neurot.2025.e00754

Figure Lengend Snippet: Proposed molecular mechanism linking DNMT1 loss to progressive LINE-1 hypomethylation in gliomas along with alterations in repressive histone marks (H4K20me3, H3K9me3) affecting chromatin structure and inducing retrotransposon activation that leads to genomic instability and tumor progression.

Article Snippet: Incubation of all the sections was performed overnight with monoclonal primary DNMT1 antibody (sc-271729, Santa Cruz Biotechnology, Dallas, USA, 1:100) or H4K20me3 (Ab 78517, Abcam, Cambridge, UK, 1:300) or H3K9me3 (CMA 308, Merck Millipore, MA, USA, 1:100).

Techniques: Activation Assay

Journal: Frontiers in Behavioral Neuroscience

Article Title: Mild traumatic brain injury increases vulnerability to post-traumatic stress disorder in rats and the possible role of hippocampal DNA methylation

doi: 10.3389/fnbeh.2025.1539028

Figure Lengend Snippet: The effect of mTBI and SPS on hippocampal DNMT1 and DNMT3b protein levels in rats. (A) Representative western blots of DNMT1, DNMT3b, and β-actin. (B) The level of DNMT1 relative to β-actin. (C) The level of DNMT3b relative to β-actin. The order of the western blot panels in (B) and (C) is the same as that in (A) . Values are presented as means ± SEM of 8 rats per group. CON, control; PTSD, post-traumatic stress disorder; mTBI, mild traumatic brain injury; SPS, single prolonged stress. *** p < 0.001, * p < 0.05.

Article Snippet: The membranes were blocked with 5% nonfat milk at room temperature for 1 h and then incubated at 4°C overnight with primary antibodies targeting DNMT1 (1:1,000; Cell Signaling, Boston, MA, USA), DNMT3b (1:1,000; Abcam, Cambridge, UK), and β -actin (1:500; Abcam).

Techniques: Western Blot, Control

Journal: Nucleic acids research

Article Title: Rescuing DNMT1 fails to fully reverse the molecular and functional repercussions of its loss in mouse embryonic stem cells.

doi: 10.1093/nar/gkaf130

Figure Lengend Snippet: Figure 1. Dnmt1 inactivation and rescue permanently reduces CpG methylation levels across genomic contexts, with over 3.2 million permanently h ypometh ylated single CpGs. ( A ) Schematic diagram depicting the Dnmt1 tet / tet mESC model and molecular and cellular analyses performed. Created in BioRender: Dupas, T. (2025) https:// BioRender.com/ a16u385 . ( B ) Pairwise comparisons of single CpG methylation levels in Dnmt1 CTL , Dnmt1 INV , and Dnmt1 RES (EM-seq: n = 3 per condition) relative to genomic location using the Wilcoxon rank sum test with Benjamini–Hochberg P -value adjustment. **** P -value ≤0.0 0 01. ( C and D ) Differential methylation levels of genome-wide single CpGs in Dnmt1 INV and Dnmt1 RES versus Dnmt1 CTL . Hypermethylated: increase ≥20%. Hypomethylated: decrease ≥20%. ( E ) Methylation alteration patterns of single CpGs during Dnmt1 inactivation and rescue determined by differential states in panels (C) and (D). Temporary: hypo / hyper in Dnmt1 INV and stable in Dnmt1 RES . Permanent: hypo / hyper in both Dnmt1 INV and Dnmt1 RES . Dynamic: h ypo / h yper in Dnmt1 INV and the opposite in Dnmt1 RES . Latent: stable in Dnmt1 INV and h ypo / h yper in Dnmt1 RES . Permanently stable: stable in both Dnmt1 INV and Dnmt1 RES . See also Supplementary Fig. S8 A.

Article Snippet: The membrane was hen incubated under soft agitation overnight at 4 ◦C with he primary antibodies for DNMT1 (Cell Signaling Technolgy, 5032) and β-actin (Cell Signaling Technology, 4970) diuted at 1 / 1000 in 5% milk.

Techniques: CpG Methylation Assay, Methylation, Genome Wide

Journal: Nucleic acids research

Article Title: Rescuing DNMT1 fails to fully reverse the molecular and functional repercussions of its loss in mouse embryonic stem cells.

doi: 10.1093/nar/gkaf130

Figure Lengend Snippet: Figure 2. Dnmt1 inactivation and rescue in mESCs alters genome-wide histone modification landscapes. ( A and B ) DHMs for H3K4me3, H3K27ac, H3K4me1, H3K27me3, and H3K9me3 in Dnmt1 INV and Dnmt1 RES versus Dnmt1 CTL measured by ChIP-seq ( n = 2 per condition) and determined by a P -value ≤0.05 and log 2 fold change ≤−0.6 or ≥0.6 using the MAnorm2 software. ( C ) Alteration patterns of DHMs for each histone modification throughout Dnmt1 inactivation and rescue determined by their differential state in panels (A) and (B). Temporary: decrease / increase in Dnmt1 INV and stable in Dnmt1 RES . Permanent: decrease / increase in both Dnmt1 INV and Dnmt1 RES . Dynamic: decrease / increase in Dnmt1 INV and the opposite in Dnmt1 RES . Latent: stable in Dnmt1 INV and decrease / increase in Dnmt1 RES . ( D and E ) Frequency of DHM alteration patterns for Dnmt1 INV and Dnmt1 RES , respectively, representing the number of DHMs in each pattern divided by the total number of DHMs. See also Supplementary Fig. S1 .

Article Snippet: The membrane was hen incubated under soft agitation overnight at 4 ◦C with he primary antibodies for DNMT1 (Cell Signaling Technolgy, 5032) and β-actin (Cell Signaling Technology, 4970) diuted at 1 / 1000 in 5% milk.

Techniques: Genome Wide, Modification, ChIP-sequencing, Software

Journal: Nucleic acids research

Article Title: Rescuing DNMT1 fails to fully reverse the molecular and functional repercussions of its loss in mouse embryonic stem cells.

doi: 10.1093/nar/gkaf130

Figure Lengend Snippet: Figure 3. Permanent CpG h ypometh ylation is associated with specific genomic location contexts and histone modification alteration patterns. ( A ) Frequency of permanently h ypometh ylated CpGs per genomic location context. ( B ) Pearson’s chi-squared test associating the frequency of permanently h ypometh ylated CpGs with genomic location contexts. ( C ) Frequency of permanently hypomethylated CpGs in DHMs categorized into alteration patterns identified in Fig. 2 C. ( D ) P earson ’s chi-squared test associating the frequency of permanently h ypometh ylated CpGs with DHM alteration patterns. Frequency of permanently h ypometh ylated CpGs represents the number of permanently h ypometh ylated CpGs divided by the number of meth ylated (meth ylation le v el ≥20%) CpGs in Dnmt1 CTL . P earson ’s residual ≥2 indicates positive, < 2 but > −2 indicates neutral and ≤−2 indicates negative association. See also Supplementary Tables S1 and S2 and Supplementary Fig. S2 .

Article Snippet: The membrane was hen incubated under soft agitation overnight at 4 ◦C with he primary antibodies for DNMT1 (Cell Signaling Technolgy, 5032) and β-actin (Cell Signaling Technology, 4970) diuted at 1 / 1000 in 5% milk.

Techniques: Modification

Journal: Nucleic acids research

Article Title: Rescuing DNMT1 fails to fully reverse the molecular and functional repercussions of its loss in mouse embryonic stem cells.

doi: 10.1093/nar/gkaf130

Figure Lengend Snippet: Figure 4. Analysis of known imprinted regions enables the identification of 20 regions with imprinted-like epigenetic and regulatory signatures. ( A ) Selection of 15 known imprinted regions based on four criteria: permanently hypomethylated CpGs ≥1, permanent mean CpG hypomethylation ≥10%, o v erlapping permanently decreased H3K9me3 DHMs, and located within H3K9me3 broad domains ( ≥30 0 0 bp) in Dnmt1 CTL . ( B ) Workflow for identifying imprinted-like regions among mESC allele-specific methylated regions based on the four criteria in panel (A). Created in BioRender: Dupas, T. (2025) https:// BioRender.com/ r69z354 . ( C ) Z-score normalization of epigenetic modification le v els in Dnmt1 CTL , Dnmt1 INV , and Dnmt1 RES f or selected known imprinted regions and imprinted-like regions. ( D and E ) Mean CpG methylation levels in oocytes versus sperm for selected known imprinted regions and imprinted-like regions, respectively. ( F ) ZFP57 DNA binding motif MA1583.1. ( G ) Enrichment of ZFP57 DNA binding motif MA1583.1 in selected known imprinted regions and imprinted-like regions. ( H ) Genomic signal tracks for H19 , Peg13–Trappc9 , and Zrsr1–Commd1 imprinted regions and Zfp1 3 , Rnf21 6 upstream , and Zfp668 imprinted-like regions showing CpG methylation in gametes and Dnmt1 tet / tet mESCs, H3K9me3 in Dnmt1 tet / tet

Article Snippet: The membrane was hen incubated under soft agitation overnight at 4 ◦C with he primary antibodies for DNMT1 (Cell Signaling Technolgy, 5032) and β-actin (Cell Signaling Technology, 4970) diuted at 1 / 1000 in 5% milk.

Techniques: Selection, Methylation, Modification, CpG Methylation Assay, Binding Assay

Journal: Nucleic acids research

Article Title: Rescuing DNMT1 fails to fully reverse the molecular and functional repercussions of its loss in mouse embryonic stem cells.

doi: 10.1093/nar/gkaf130

Figure Lengend Snippet: Figure 5. Permanent derepression of MERVL and MT2 LTRs may be linked to gene transcript chimerism. ( A ) Frequency of permanently h ypometh ylated CpGs in the most abundant LTR families and in high-occupancy LTRs (genomic occurrences ≥500). ( B and C ) Change in mean CpG methylation levels (EM-seq) and log 2 fold change of total RNA-seq normalized counts, respectively, of high-occupancy LTRs in Dnmt1 INV and Dnmt1 RES versus Dnmt1 CTL

Article Snippet: The membrane was hen incubated under soft agitation overnight at 4 ◦C with he primary antibodies for DNMT1 (Cell Signaling Technolgy, 5032) and β-actin (Cell Signaling Technology, 4970) diuted at 1 / 1000 in 5% milk.

Techniques: CpG Methylation Assay, RNA Sequencing

Journal: Nucleic acids research

Article Title: Rescuing DNMT1 fails to fully reverse the molecular and functional repercussions of its loss in mouse embryonic stem cells.

doi: 10.1093/nar/gkaf130

Figure Lengend Snippet: Figure 6. Gene expression dysregulation, genomic inst abilit y, and altered cellular processes. ( A and B ) Differentially expressed genes in Dnmt1 INV and Dnmt1 RES versus Dnmt1 CTL determined by an adjusted P -value ≤0.05 and log 2 fold change ≤−0.6 or ≥0.6 using mRNA sequencing ( n = 3 per

Article Snippet: The membrane was hen incubated under soft agitation overnight at 4 ◦C with he primary antibodies for DNMT1 (Cell Signaling Technolgy, 5032) and β-actin (Cell Signaling Technology, 4970) diuted at 1 / 1000 in 5% milk.

Techniques: Gene Expression, Sequencing

Journal: Nucleic acids research

Article Title: Rescuing DNMT1 fails to fully reverse the molecular and functional repercussions of its loss in mouse embryonic stem cells.

doi: 10.1093/nar/gkaf130

Figure Lengend Snippet: Figure 7. Lasting effects on gene expression patterns upon differentiation to embryonic germ layers. ( A ) Schematic diagram depicting Dnmt1 CTL and Dnmt1 RES differentiation into germ la y er monola y ers ( n = 3 per differentiation). Created in BioR ender: Dupas, T. (2025) https://BioR ender.com/y05v644 . ( B ) Differential gene expression in Dnmt1 RES versus Dnmt1 CTL endoderm, mesoderm and ectoderm determined by an adjusted P -value ≤0.05 and log 2 fold change ≤−0.6 or ≥0.6 using mRNA sequencing ( n = 3 per differentiation) analyzed with the DESeq2 software. ( C ) GSEA for down- and upregulated genes in Dnmt1 RES germ la y ers. ( D ) Total number of differentially expressed genes in Dnmt1 RES cell types. ( E and F ) Overlap of downregulated and upregulated genes among Dnmt1 RES cell types. See also Supplementary Table S6 and Supplementary Fig. S7 .

Article Snippet: The membrane was hen incubated under soft agitation overnight at 4 ◦C with he primary antibodies for DNMT1 (Cell Signaling Technolgy, 5032) and β-actin (Cell Signaling Technology, 4970) diuted at 1 / 1000 in 5% milk.

Techniques: Gene Expression, Sequencing, Software

Journal: Nucleic acids research

Article Title: Rescuing DNMT1 fails to fully reverse the molecular and functional repercussions of its loss in mouse embryonic stem cells.

doi: 10.1093/nar/gkaf130

Figure Lengend Snippet: Figure 8. Delaying Dnmt1 rescue until after differentiation initiation worsens molecular and cellular outcomes. ( A ) Schematic diagram depicting embryoid body formation including four experimental conditions: Dnmt1 -control, Dnmt1 -inactive, Dnmt1 -rescue, and post-differentiation Dnmt1 -rescue. Embryoid bodies ( n = 192 per condition) were derived from the same Dnmt1 tet / tet mESC culture. Created in BioRender: Dupas, T. (2025) https:// BioRender.com/ f72a244 . ( B ) Bright-field microscopy images of one embryoid body per condition taken on days 3, 5, 7, and 10. ( C ) Size and ( D ) circularity of embryoid bodies on days 2, 5, and 10 determined by measuring their surface area and perimeter on bright-field microscopy images using the ImageJ software. Daily images were taken of the same 20 embryoid bodies per condition. Surface area was used as an indicator of size and circularity was calculated with the formula 4 π × Area / Perimeter 2 . ( E ) Principal component analysis of gene expression measured on day 5 and day 10 by mRNA sequencing ( n = 3 per condition with pooled embryoid bodies). A greater divergence in gene expression profiles is represented by the distance between samples along the x -axis (PC1), compared to the y -axis (PC2). ( F ) Number of differentially expressed genes in Dnmt1 -inactive, Dnmt1 -rescue, and post-differentiation Dnmt1 -rescue embryoid bodies versus Dnmt1 -control embryoid bodies on day 5 and day 10 determined by an adjusted P -value ≤0.05 and log2 fold change ≤−0.6 or ≥0.6 using mRNA sequencing analyzed with the DESeq2 software. ( G ) Estimated proportion of ExE endoderm, epiblast (pluripotent), primitive streak (mesendoderm), and neurectoderm cell types in embryoid bodies on days 5 and 10 determined by decon v oluting ra w counts using single-cell data from mouse gastrulation embry os (embry onic da y 7.75). All pairwise comparisons w ere conducted using one-w a y ANO V A with Tuk e y’s HSD test and P -v alue adjustment. ns P -v alue > 0.05, * P -v alue ≤0.05, ** P -v alue ≤0.01, *** P -v alue ≤0.001, **** P -value ≤0.0 0 01. See also Supplementary Fig. S8 .

Article Snippet: The membrane was hen incubated under soft agitation overnight at 4 ◦C with he primary antibodies for DNMT1 (Cell Signaling Technolgy, 5032) and β-actin (Cell Signaling Technology, 4970) diuted at 1 / 1000 in 5% milk.

Techniques: Control, Derivative Assay, Microscopy, Software, Gene Expression, Sequencing

Journal: Scientific reports

Article Title: Milk derived extracellular vesicle uptake in human microglia regulates the DNA methylation machinery : Short title: milk-derived extracellular vesicles and the epigenetic machinery.

doi: 10.1038/s41598-024-79724-1

Figure Lengend Snippet: Fig. 4. The effects of milk-derived extracellular vesicles (MEVs) supplementation on baseline homeostatic human microglia clone 3 (HMC3) cells at 12 h post-supplementation. DNMT1 and miR-148-5P transcript levels (a), DNMT1 protein abundance (b) and DNMT enzymatic activity (c). Spearman correlations for DNMT1 levels and miR-148-5P levels (d), and DNMT enzymatic activity and DNMT1 protein level (e). * p < 0.05, ** p < 0.01, **** p < 0.0001. Error bars represent the Standard Error of Means (± SEM).

Article Snippet: The membranes were incubated with DNMT1 primary antibody (Proteintech; 24206-I-AP, 1:500, v: v, 1X TBST) overnight at 4 °C on a rocker.

Techniques: Derivative Assay, Quantitative Proteomics, Activity Assay

Journal: Scientific reports

Article Title: Milk derived extracellular vesicle uptake in human microglia regulates the DNA methylation machinery : Short title: milk-derived extracellular vesicles and the epigenetic machinery.

doi: 10.1038/s41598-024-79724-1

Figure Lengend Snippet: Fig. 6. The effects of milk-derived extracellular vesicles (MEVs) on IFN-γ primed human microglia clone 3 (HMC3) cells at 12 h post-supplementation. DNMT1 and miR-148a-5P transcript levels (a), DNMT1 protein abundance (b), and DNMT enzymatic activity (c). Spearman correlations for DNMT1 levels and miR-148-5P levels (p < 0.05) (d), and DNMT enzymatic activity and DNMT1 protein level (p < 0.05) (e). P-MEV indicates primed cells that received MEVs. P-Ctrl is primed control cells. ** p < 0.01, **** p < 0.0001. Error bars represent the Standard Error of Means (± SEM).

Article Snippet: The membranes were incubated with DNMT1 primary antibody (Proteintech; 24206-I-AP, 1:500, v: v, 1X TBST) overnight at 4 °C on a rocker.

Techniques: Derivative Assay, Quantitative Proteomics, Activity Assay, Control

Journal: Scientific reports

Article Title: Milk derived extracellular vesicle uptake in human microglia regulates the DNA methylation machinery : Short title: milk-derived extracellular vesicles and the epigenetic machinery.

doi: 10.1038/s41598-024-79724-1

Figure Lengend Snippet: Fig. 7. The effects of milk-derived extracellular vesicles (MEVs) on primed human microglia clone 3 (HMC3) cells 9 h post-supplementation. DNMT1 and miR-148a-5P transcript levels (a), DNMT1 protein abundance (b), and DNMT enzymatic activity (c). Spearman correlations for DNMT1 levels relative to miR-148a-5P levels (p ≤ 0.05) (d), and DNMT enzymatic activity relative to DNMT1 protein level (A.U) (e). P-MEV indicates primed cells that received MEVs. P-Ctrl is primed control cells. ** p < 0.01, **** p < 0.0001. Error bars represent the Standard Error of Means (± SEM).

Article Snippet: The membranes were incubated with DNMT1 primary antibody (Proteintech; 24206-I-AP, 1:500, v: v, 1X TBST) overnight at 4 °C on a rocker.

Techniques: Derivative Assay, Quantitative Proteomics, Activity Assay, Control