chip grade antibodies anti h3k4me3  (Cell Signaling Technology Inc)

Journal: Life Science Alliance

Article Title: Dectin-1 epigenetic reprogramming rescues senescent-like Treg function in allergic asthma

doi: 10.26508/lsa.202503552

Figure Lengend Snippet: (A) Binding of KQS-1 to purified human Tregs. Cells were incubated with KQS-1 and analyzed by flow cytometry to determine the proportion of KQS-1–positive cells. Representative gating strategies are shown in . (B, C, D) Raf-1 and ROS dependence of KQS-1–primed Treg responses. Tregs were treated with KQS-1 alone or in combination with the Raf-1 inhibitor GW5074 (10 μM) or the ROS scavenger N-acetylcysteine (NAC, 5 mM). (B) H3K4me3 enrichment at the FOXP3 promoter assessed by ChIP–qPCR and expressed in arbitrary units (a.u.). (C) In vitro suppressive capacity of treated Tregs. The dashed line indicates the mean suppression achieved by KQS-1–treated cells. Representative flow cytometry plots are provided in . (D) TGF-β protein levels in culture supernatants under the indicated conditions. (C) Data are presented as the mean ± SD, with each dot representing an individual donor (n = 10 biologically independent replicates for panel (C)). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. **** P < 0.0001.

Article Snippet: ChIP-grade antibodies anti-H3K4me3 (clone: C42D8) and anti-H3K27ac (clone: D8L3M) (Cell Signaling Technology); ChIP assay kit (Millipore); and Illumina Infinium HumanMethylation450K BeadChip (Illumina) were used to perform ChIP–qPCR to analyze epigenetic changes at specific genes.

Techniques: Binding Assay, Purification, Incubation, Flow Cytometry, ChIP-qPCR, In Vitro

Journal: Life Science Alliance

Article Title: Dectin-1 epigenetic reprogramming rescues senescent-like Treg function in allergic asthma

doi: 10.26508/lsa.202503552

Figure Lengend Snippet: (A) ChIP–qPCR analysis of active histone marks at key Treg signature loci. Enrichment of H3K4me3 at the FOXP3 promoter and IL-10 locus, as well as global H3K27ac levels, was assessed in KQS-1–treated and control cells. (B) ATAC-seq volcano plot comparing chromatin accessibility in KQS-1–treated versus untreated Tregs, highlighting newly accessible genomic regions. (C) Genomic annotation of KQS-1–induced accessible regions, categorized as promoters (±3 kb of the transcription start site), enhancers (distal regulatory elements), or intergenic regions. Distribution of accessibility changes across genomic features is shown. (D) Chromatin accessibility at IL-10 and FOXP3 promoter regions. Upper panels show representative ATAC-seq signal tracks; lower panels present quantification of accessibility changes after KQS-1 treatment. (E) Gene ontology (GO) enrichment analysis of genes proximal to KQS-1–induced accessible regions, illustrating enrichment of immune regulatory and T cell–associated pathways. (F) DNA methylation profiling of regulatory regions using the Illumina 450K array. Lollipop plots depict changes in methylation (Δβ) between KQS-1–treated and untreated Tregs, highlighting focal hypomethylation at FOXP3 and IL-10 regulatory CpG sites. ChIP–qPCR data are presented as the mean ± SD (n = 8 biological replicates), with individual points representing replicates. ATAC-seq analysis identified 1,306 newly accessible regions (FDR < 0.05). For genomic distribution analysis, box plots show median and interquartile range (IQR), with individual dots representing peaks. DNA methylation data are shown as mean Δβ with 95% confidence intervals. Statistical significance was determined using paired or unpaired t tests, one-way ANOVA with Tukey’s post hoc test, and the Bonferroni correction for multiple comparisons, as appropriate. ** P < 0.01, *** P < 0.001, **** P < 0.0001; ns, not significant.

Article Snippet: ChIP-grade antibodies anti-H3K4me3 (clone: C42D8) and anti-H3K27ac (clone: D8L3M) (Cell Signaling Technology); ChIP assay kit (Millipore); and Illumina Infinium HumanMethylation450K BeadChip (Illumina) were used to perform ChIP–qPCR to analyze epigenetic changes at specific genes.

Techniques: ChIP-qPCR, Control, DNA Methylation Assay, Methylation

Journal: Life Science Alliance

Article Title: Dectin-1 epigenetic reprogramming rescues senescent-like Treg function in allergic asthma

doi: 10.26508/lsa.202503552

Figure Lengend Snippet: Gain-of-function analysis in Dectin-1-low Tregs after lentiviral transduction with Dectin-1 (Dectin-1 OE) or empty vector control. (A, B) Flow cytometry analysis showing a significant increase in KQS-1 binding (MFI) in Dectin-1 OE cells compared with controls (A), accompanied by restored H3K4me3 epigenetic marks at the FOXP3 promoter region as measured by ChIP–qPCR (B). (C) Suppressive efficiency of Tregs against responder T-cell proliferation, demonstrating enhanced inhibitory capacity upon Dectin-1 restoration. Data are presented as box-and-whisker plots with individual samples shown as dots (n = 5 per group). Statistical significance was determined by an unpaired t test; * P < 0.05, ** P < 0.01.

Article Snippet: ChIP-grade antibodies anti-H3K4me3 (clone: C42D8) and anti-H3K27ac (clone: D8L3M) (Cell Signaling Technology); ChIP assay kit (Millipore); and Illumina Infinium HumanMethylation450K BeadChip (Illumina) were used to perform ChIP–qPCR to analyze epigenetic changes at specific genes.

Techniques: Transduction, Plasmid Preparation, Control, Flow Cytometry, Binding Assay, ChIP-qPCR, Whisker Assay

Journal: The Journal of Biological Chemistry

Article Title: Unraveling MLL1-fusion leukemia: Epigenetic revelations from an iPS cell point mutation

doi: 10.1016/j.jbc.2024.107825

Figure Lengend Snippet: Loss of MLL1 enzymatic activity results in a global increase and redistribution of H3K4 trimethylation. A , immunoblot analysis of H3K4me1-3 levels in WT and MT hIPS cells. Total H3 was used as the loading control. B , immunofluorescence staining with anti-H3K4me3 antibody of WT and MT hIPS cells. C , ChIP-seq heat maps and profiles were generated from H3K4me3–control IgG signals and plotted over transcription start sites (TSS±5kb) for WT and MT hIPS cells. D , pie charts for H3K4me3 enrichment versus IgG in annotated genomic regions for WT and MT hIPS cells. E , metagene plots for H3K4me3 ChIP-seq signal (-IgG) at WT MACS2 peak summits ( top row ) and at MT1 + MT2 MACS2 peak summits ( bottom row). F , KEGG enrichment analysis of top canonical pathways represented in overlapping ChIP-Seq peaks from MT1 and MT2 clones normalized to WT. Log 10 of enrichment p -values ( p < 0.05) for each pathway is represented on the x -axis. G – I , integrated genomics viewer (IGV) browser snapshots comparing H3K4me3–IgG ChIP-seq enrichment signals between WT and MT hIPS cells at the Meis1 promoter ( G ), HoxA13-HOTTIP promoter ( H ), the Zeb1 promoter ( I ). ChIP-seq, chromatin immunoprecipitation followed by sequencing; H3K4, H3 lysine 4; H3K4me, H3 lysine 4 methylation; hIPS, human-induced pluripotent stem; IgG, immunoglobulin G; KEGG, Kyoto Encyclopedia for Genes and Genomes.

Article Snippet: For H3K4me3, 1 μg of Rabbit anti-H3K4me3 ChIP grade antibody (EpiCypher, 13–0041), which has been SNAP-ChIP validated for H3K4me3 specificity , was used for the immunoprecipitation.

Techniques: Activity Assay, Western Blot, Control, Immunofluorescence, Staining, ChIP-sequencing, Generated, Clone Assay, Chromatin Immunoprecipitation, Sequencing, Methylation

Journal: The Journal of Biological Chemistry

Article Title: Unraveling MLL1-fusion leukemia: Epigenetic revelations from an iPS cell point mutation

doi: 10.1016/j.jbc.2024.107825

Figure Lengend Snippet: Model for the role of MLL1 histone methyltransferase activity in normal and leukemic stem cells . A , WT MLL1-catalyzed H3K4 dimethylation (H3K4me2) limits the spread of the gene-activating H3K4 trimethylation (H3K4me3) mark catalyzed by SETd1a in WT iPS cells. B , homozygous loss-of-function point mutation of MLL1 prevents deposition of the H3K4me2 mark at MLL1 target genes. This allows spreading of the SETd1a-catalyzed H3K4me3 mark and activation of LSC maintenance genes Hoxa9, HoxA10, Meis1, and so on. C , model for MLL1 F -induced leukemogenesis. In this model, the MLL1 F protein acts in a dominant negative manner by interaction with or displacement of WT MLL1 and prevents H3K4me2 methylation at MLL1 target genes. As in the homozygous loss-of-function point mutant cells ( B ), lack of MLL1 catalytic activity allows spreading of the SETd1a-catalyzed H3K4me3 mark to MLL1 target gene promoters resulting in expression of LSC maintenance genes. (Created with BioRender.com ). H3K4, H3 lysine 4; H3K4me, H3 lysine 4 methylation; iPS, induced pluripotent stem; LSC, leukemic stem cell; MLL, mixed lineage leukemia; MLL1 F , MLL1-fusion protein.

Article Snippet: For H3K4me3, 1 μg of Rabbit anti-H3K4me3 ChIP grade antibody (EpiCypher, 13–0041), which has been SNAP-ChIP validated for H3K4me3 specificity , was used for the immunoprecipitation.

Techniques: Activity Assay, Mutagenesis, Activation Assay, Dominant Negative Mutation, Methylation, Expressing

Journal: Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy

Article Title: H3K4me3 remodeling induced acquired resistance through O-GlcNAc transferase ☆

doi: 10.1016/j.drup.2023.100993

Figure Lengend Snippet: a, Immunoblotting of H3K4me3 at the individual cell states in WM164, A375, A549 and H1975 cells. Histone 3 were used as loading control. Representative images of independent experiments are shown (n ≥ 2). b, Global H3K4me3 ChIP-seq profiles of parent, IDTC colony and resistant cells, replicates (all n = 2) were combined for visualization. c , Over-representation analysis of genes annotated to significantly (FDR<5%) differentially marked proximal H3K4me3 peaks against Reactome pathways database of parent compared to IDTC colony or resistant. The top 10 most significantly enriched pathways are shown. d, Scatter plot showing log2 fold change of differentially marked proximal H3K4me3 peaks (colony vs parental) as determined by ChIP-seq and mRNA expression levels of differentially expressed genes (colony vs parental) as determined by microarray. Shown are all genes that were significantly differentially expressed (p ≤ 0.05, absolute log2FC ≥ 0.5) in both datasets. The slope is indicated by the red line. e, Venn diagrams comparing annotated genes with increased ( upper panel ) and decreased ( lower panel ) H3K4me3 in IDTC colony (Col) compared to parent (P) and resistant (R) compared to parent (P).

Article Snippet: ChIP-grade H3K4me3 (Active Motif Cat: 39159), O-GlcNAc antibody (Active Motif Cat: 61454) or rabbit IgG (Cell Signaling #2729) was used to perform the pulldown.

Techniques: Western Blot, Control, ChIP-sequencing, Expressing, Microarray

Journal: Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy

Article Title: H3K4me3 remodeling induced acquired resistance through O-GlcNAc transferase ☆

doi: 10.1016/j.drup.2023.100993

Figure Lengend Snippet: a and b , Transcription factor binding motifs of proximal H3K4me3 peaks identified in IDTC colony and resistant cells ( a ) or significantly (FDR<5%) differentially marked proximal H3K4me3 peaks of all conditions ( b ). Top 20 most significantly discovered motifs were compared and commonly found sequence logos are shown. c and d, Venn diagram comparing annotated CpG islands from the USC genome browser for hg19 with significantly (FDR<5%) differentially marked proximal H3K4me3 peaks (ΔH3K4me3) of parent (P) vs IDTC colony (Col) ( c middle panel) or parent (P) vs resistant (R) ( d middle panel). Top 5 transcription factor binding motifs identified in significantly differentially marked proximal H3K4me3 peaks (lower panel) and significantly differentially marked proximal H3K4me3 peaks that also overlap with CpG islands (upper panel) are shown. e, Scatter plot of log2FC of the differentially marked proximal H3K4me3 peaks of parent vs IDTC colony as determined by ChIP-seq and log2FC of mRNA levels as determined by RT-qPCR of 21 resistance-associated genes. Genes with significant changes in their mRNA level are indicated in red. Genes with H3K4me3 peaks overlapping with CpG islands are indicated by squares.

Article Snippet: ChIP-grade H3K4me3 (Active Motif Cat: 39159), O-GlcNAc antibody (Active Motif Cat: 61454) or rabbit IgG (Cell Signaling #2729) was used to perform the pulldown.

Techniques: Binding Assay, Sequencing, ChIP-sequencing, Quantitative RT-PCR

Journal: Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy

Article Title: H3K4me3 remodeling induced acquired resistance through O-GlcNAc transferase ☆

doi: 10.1016/j.drup.2023.100993

Figure Lengend Snippet: Immunoblotting of OGT, TET1 ( a ) and O-GlcNAc ( b ) at the individual cell states in WM164, A375 ( a only), A549 and H1975 cells. β-Actin was used as loading control. c, Immunoblotting of OGT, TET1 and O-GlcNAc in PDX tumors either untreated (control) or exposed to TAK-733 for 30 days. GAPDH was used as processing and loading control. Representative images are shown (n ≥ 2). d , Schematic representation of the hexosamine biosynthesis pathway. e , Abundance of the indicated metabolites (QC normalized data) from WM164 cells (IDTC and IDTC colony n = 6, parent and resistant n = 7) at individual cell states. P-Values were calculated using ANOVA, * ** P ≤ 0.001, * * P ≤ 0.01, * P ≤ 0.05, n.s. P > 0.05, Data are represented as mean +SD. f, Immunoprecipitation of O-GlcNAcylated proteins in WM164, A549 and H1975 parent or IDTC colony cells followed by immunoblotting for histone 3. Cell lysates were normalized to total protein concentration, and OGT was used as an IP positive control. Representative images of independent experiments are shown (n ≥ 2). g , Immunoblotting of O-GlcNAc and H3K4me3 after treatment with PLX4032 (1 μM) (45 days) or OSMI4B (10 μM) (48 h), alone or in combination (45 days). β-Actin and H3 was used as loading control. h, Transcript levels of NRG1, WNT5A, VEGFC and EGFR after treatment with PLX4032 (1 μM) (45 days) or OSMI4B (10 μM) (48 h), alone or in combination (45 days). P-Values were calculated using ANOVA, * ** P ≤ 0.001, * * P ≤ 0.01, * P ≤ 0.05, n.s. P > 0.05, Data are represented as mean +SD.

Article Snippet: ChIP-grade H3K4me3 (Active Motif Cat: 39159), O-GlcNAc antibody (Active Motif Cat: 61454) or rabbit IgG (Cell Signaling #2729) was used to perform the pulldown.

Techniques: Western Blot, Control, Immunoprecipitation, Protein Concentration, Positive Control

Journal: Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy

Article Title: H3K4me3 remodeling induced acquired resistance through O-GlcNAc transferase ☆

doi: 10.1016/j.drup.2023.100993

Figure Lengend Snippet: a , Crystal violet staining of shOGT and shControl WM164 and A549 following exposure to dabrafenib (50nM) or docetaxel (5nM), respectively, for 45 days (left panels). Immunoblotting of OGT in WM164 and A549 (right panels) following transfection with shRNA targeting OGT. β-Actin was used as loading control. Representative images of independent experiments are shown (n≥2). b , Same as (a) with shTET1. Representative images of independent experiments are shown (n≥2). c , Tumor growth of WM164 shControl, shOGT and shTET1 cells injected into flanks of immunocompromised mice and treated with vehicle or dabrafenib (10 mg/kg)/trametinib (0.1 mg/kg), (n=6 per group). P-Values were calculated using t-tests corrected for multiple comparisons, *** P ≤ 0.001, Data are represented as mean ±SD. d , Immunoblotting of OGT, TET1 and O-GlcNAc from isolated tumor samples treated with vehicle (11 days) or dabrafenib (10 mg/kg)/trametinib (0.1 mg/kg) (treated, 27 days). β-Actin was used as loading control. Samples correspond to e and representative images of independent experiments are shown (n≥2). e , Immunofluorescence of H3K4me3 (red) combined with Hoechst nuclear staining (blue) of tumor samples treated with vehicle or dabrafenib (10 mg/kg)/trametinib (0.1 mg/kg). Representative images of independent experiments are shown (n≥3). f , Tumor growth of A549 shControl and shOGT cells injected into flanks of immunocompromised mice and treated with vehicle or docetaxel (15 mg/kg) (n=4 per group). P-Values were calculated using t-tests corrected for multiple comparisons, * P ≤ 0.05. Data are represented as mean ±SD.

Article Snippet: ChIP-grade H3K4me3 (Active Motif Cat: 39159), O-GlcNAc antibody (Active Motif Cat: 61454) or rabbit IgG (Cell Signaling #2729) was used to perform the pulldown.

Techniques: Staining, Western Blot, Transfection, shRNA, Control, Injection, Isolation, Immunofluorescence

Journal: Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy

Article Title: H3K4me3 remodeling induced acquired resistance through O-GlcNAc transferase ☆

doi: 10.1016/j.drup.2023.100993

Figure Lengend Snippet: a , Crystal violet staining of WM164 and A549 cells exposed to dabrafenib 50 nM or docetaxel 5 nM with or without aspirin 2 mM for the indicated time frames. b, Relative growth rate of WM164 cells exposed to the indicated drug combinations for the indicated time points using MTT assay (n = 3). P-values were calculated using one-way ANOVA, * ** P ≤ 0.001, n.s. P > 0.05, Data are represented as mean ± SD. c, Relative growth rate of WM164 parental cells compared to WM164 cells exposed to dabrafenib 50 nM and aspirin 2 mM for 235 days, followed by 21 days drug holiday and re-treatment with indicated dabrafenib concentrations for 72 h (n = 3). Data are represented as mean ± SD. d, Immunoblotting of phospho-acetyl-CoA carboxylase (p-ACC), OGT, TET1 and O-GlcNAc from WM164 and A549 cells following indicated treatments. β-Actin was used as loading control. Representative images of independent experiments are shown (n ≥ 2). e and f, Immunofluorescence staining of H3K4me3 (red) combined with Hoechst nuclear staining (blue) in WM164 shControl or shAMPK cells with indicated treatments. Representative images of independent experiments are shown (n ≥ 3). g, Tumor growth of WM164 cells that were treated in vitro with either dabrafenib 50 nM or dabrafenib 50 nM combined with aspirin 2 mM for 50 days before injection into immunocompromised mice (n = 3). P-values were calculated using t-tests corrected for multiple comparisons, * ** P ≤ 0.001, Data are represented as mean ± SD. h, Immunoblotting of OGT, TET1 and O-GlcNAc from isolated tumor samples treated with vehicle, aspirin (100 mg/kg), dabrafenib (10 mg/kg)/trametinib (0.1 mg/kg) or the combination. GAPDH was used as loading control. Representative images of independent experiments are shown (n ≥ 2). i, Immunofluorescence staining of H3K4me3, OGT and TET1 (all red) combined with Hoechst nuclear staining (blue) of tumor samples treated with vehicle, aspirin (100 mg/kg) dabrafenib (10 mg/kg)/trametinib (0.1 mg/kg) or the combination. Representative images of independent experiments are shown (n ≥ 3).

Article Snippet: ChIP-grade H3K4me3 (Active Motif Cat: 39159), O-GlcNAc antibody (Active Motif Cat: 61454) or rabbit IgG (Cell Signaling #2729) was used to perform the pulldown.

Techniques: Staining, MTT Assay, Western Blot, Control, Immunofluorescence, In Vitro, Injection, Isolation