H3k4me3

Manufactured by Cell Signaling Technology

Sourced in United States, United Kingdom, China

H3K4me3 is an antibody that specifically recognizes the trimethylated form of histone H3 at lysine 4. Histone modifications, such as methylation, play a crucial role in regulating gene expression and chromatin structure. H3K4me3 is associated with active transcription and is commonly used as a marker for actively transcribed regions of the genome.

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Anti-H3K4me3 (57 citations) Rabbit anti-H3K4me3 (10 citations) H3K4me3 antibody (8 citations) Anti-H3K4me3 antibody (4 citations) H3K4me3 C42D8 (3 citations) Anti-H3K4me3 (9751s; (2 citations) Rabbit α H3K4me3 (2 citations) Anti-H3K4me3 (C42D8; (2 citations) Rabbit anti-H3K4me3 antibody (2 citations) α-H3K4me3 (2 citations)

106 protocols using h3k4me3

TROP2 Expression in Colorectal Tumors

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FFPE colorectal tumor samples were obtained from excision biopsies as described previously^{14 (link)}. Tissue blocks were assembled. Slices (2 µM thick) were stained with HE, TROP2 (1:2000, rabbit IgG, ab214488, Abcam, Cambridge, UK), and H3K4me3 (1:1000, rabbit IgG, #9751, Cell Signaling, Danvers, USA). The samples were scored for TROP2 expression by a pathologist (KEW). The total immunostaining score (0–300) consists of the intensity (0–3) and the percentage of positive cells (0–100%). Using the median of 90, the tumor samples were categorized into a TROP2 high-expressing group (> 90) and a TROP2 low-expressing group (< 90).

Gehring A., Huebner K., Rani H., Erlenbach-Wuensch K., Merkel S., Mahadevan V., Grutzmann R., Hartmann A, & Schneider-Stock R. (2024). DNA demethylation and tri-methylation of H3K4 at the TACSTD2 promoter are complementary players for TROP2 regulation in colorectal cancer cells. Scientific Reports, 14, 2683.

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Examining EPB41L4A-AS1 RNA Interactions

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Different fractions of EPB41L4A-AS1 were constructed with the pcDNA3.1 plasmid containing the T7 promoter. The RNA pulldown assay was performed following the protocol of the Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher Scientific, USA). Antibodies, including SET1A and H3K4me3, were purchased from Cell Signaling Technology.

Zhu Y., Liu Q., Liao M., Diao L., Wu T., Liao W., Wang Z., Li B., Zhang S., Wang S., Xie W., Jiang Y., Xu N., Zeng Y., Yang B.B, & Zhang Y. (2019). Overexpression of lncRNA EPB41L4A-AS1 Induces Metabolic Reprogramming in Trophoblast Cells and Placenta Tissue of Miscarriage. Molecular Therapy. Nucleic Acids, 18, 518-532.

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Western Blot Analysis of Stress Responses

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Protein lysates were prepared using RIPA lysis buffer and separated on 4–12% NuPAGE gels (Invitrogen), transferred onto a nitrocellulose membrane (Millipore) and incubated with the following antibodies: ACTIN (ab6276) and NRF2 (EP1808Y) mAb (both Abcam), NQO1 (HPA007308) and PHGDH (HPA021241) (both Sigma), PSAT1 (PA5-22124, Pierce), and ATF4 (11815, Cell Signaling). Alternatively, nuclear extracts were prepared as described³¹. Histone extracts were prepared with the Histone Extraction Kit according to the manufacturer’s instructions (Abcam, ab113476). Histone extracts were probed with the following antibodies: H3K4me3 (9727, Cell Signaling), H3K27me3 (07-449, Millipore), total H3 (4499, Cell Signaling).

DeNicola G.M., Chen P.H., Mullarky E., Sudderth J.A., Hu Z., Wu D., Tang H., Xie Y., Asara J.M., Huffman K.E., Wistuba I.I., Minna J.D., DeBerardinis R.J, & Cantley L.C. (2015). NRF2 regulates serine biosynthesis in non-small cell lung cancer. Nature genetics, 47(12), 1475-1481.

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Antibody and Inhibitor Reagents for Assays

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Rat anti-mouse F4/80 antibody was purchased from Serotec; Rat anti-mouse CD31 was purchased from Dianova (Hamburg, Germany). Anti-actin and anti-smooth muscle actin (SMA) monoclonal antibodies were purchased from Sigma (St. Louis, MO). Antibodies directed against phopsho-AMPK alpha, phospho-p38 and phospho-JNK, histone H3 and H3K4-di (H3K4me2) and -tri (H3K4Me3) methylation, H3K27 tri-methylation and acetylated H2BK5 were purchased from Cell Signaling Technology (Beverly, MA). Anti-WDR5, anti-BiP, and anti-Ly6g antibodies were purchased from Abcam (Cambridge, UK). The selective inhibitors of LSD1 (GSK-2879552), WDR5 (OICR-9429), MKL1 (CCG-203971), CXCR2 (SB 225002), p38 (SB208530), and JNK (sp600125) were purchased from ApexBio (Boston, MA) and were dissolved in DMSO as stock solutions. DMSO (Sigma D2438) was added to the cell culture medium as control. The heparanase inhibitors H1001 and PG545 were kindly provided by HepaRx Ltd (Ness-Ziona, Israel) and Zucero Therapeutics (Darra, Queensland, Australia), respectively. Mouse CXCL2/MIP-2 Quantikine ELISA kit was purchased from R&D Systems (Minneapolis, MN). The MyD88 peptide inhibitory set was purchased from Novus Biologicals (Centennial, CO). Latent heparanase was purified from medium conditioned by CHO cells overexpressing heparanase essentially as described (25 ) and was added to cell cultures at 1 μg/ml.

Bhattacharya U., Gutter-Kapon L., Kan T., Boyango I., Barash U., Yang S.M., Liu J., Gross-Cohen M., Sanderson R.D., Shaked Y., Ilan N, & Vlodavsky I. (2019). Heparanase and chemotherapy synergize to drive macrophage activation and enhance tumor growth. Cancer research, 80(1), 57-68.

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Chromatin Immunoprecipitation Profiling of Epigenetic Factors

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Chromatin immunoprecipitation was performed as previously described 7 using antibodies against HIF‐1 (PM14), HIF‐2 (PM9) 42, 43, HIF‐1 (Novus NB‐100‐110), H3K4me1 (Millipore, #07‐436), H3K4me3 (#9751, Cell Signaling Technology), H3K27ac (#ab4729, Abcam), CTCF (#07‐729, Millipore), and RNApol2 (#sc‐899, Santa Cruz Biotechnology). Each ChIP was performed in duplicate. Libraries were prepared using PrepX Complete ILMN DNA library kit (#400076, Wafergen).

Platt J.L., Salama R., Smythies J., Choudhry H., Davies J.O., Hughes J.R., Ratcliffe P.J, & Mole D.R. (2016). Capture‐C reveals preformed chromatin interactions between HIF‐binding sites and distant promoters. EMBO Reports, 17(10), 1410-1421.

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Western Blot Analysis of Cell Cycle Regulators

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Western blots were conducted as previously described [25 (link)]. Primary antibodies specific to CDK1, CCNB1, CCNE2, BIRC5, AURKA, FOXM1, WDR5, H3K4me3 (1:1000, Cell Signalling Technology, Danvers, MA, USA), histone H3 (1:2000, Cell Signalling Technology, Danvers, MA, USA), XRCC2, MCM2, PLK1, and GAPDH (1:1000, Abcam) were used. The PVDF films were then incubated with secondary antibodies (anti-mouse or anti-rabbit, Promega, USA) and visualized using Immobilon enhanced chemiluminescence (Millipore). The full images of all Western blots are shown in Supplementary Fig. 9.

Zhang J., Zhou Q., Xie K., Cheng L., Peng S., Xie R., Liu L., Zhang Y., Dong W., Han J., Huang M., Chen Y., Lin T., Huang J, & Chen X. (2021). Targeting WD repeat domain 5 enhances chemosensitivity and inhibits proliferation and programmed death-ligand 1 expression in bladder cancer. Journal of Experimental & Clinical Cancer Research : CR, 40, 203.

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Western Blot Analysis of Mitochondrial and Epigenetic Proteins

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Western blot was performed as described^{2 (link)} using the following antibodies: TFAM (7495S, Cell Signaling Technology; sc-23588, Santa Cruz Technology), PHB2 (14085S, Cell Signaling Technology), ATP5A1 (sc-136178, Santa Cruz Technology), ATP5B (sc-16690, Santa Cruz Technology), ATP5D (ab97491, Abcam), ATP5O (ab110276, Abcam, clone 4C11C10D12), 4EBP1 (9644S, Cell Signaling Technology), phos-4EBP1 (Thr37/46) (2855S, Cell Signaling Technology), p70S6K (2708S, Cell Signaling Technology), phos-S6K (Ser235/236) (2211S, Cell Signaling Technology), GATA1 (ab47490, Abcam), GATA2 (ab22849, Abcam), TSC1 (6935, Cell Signaling Technology), TSC2 (4308, Cell Signaling Technology), H3K9ac (9649, Cell Signaling Technology), H3K27ac (8173, Cell Signaling Technology), H3K56ac (4243, Cell Signaling Technology), H4K5ac (ab51997, Abcam), H3K4me3 (9571, Cell Signaling Technology), H3K9me3 (13969, Cell Signaling Technology), H3K27me3 (9733, Cell Signaling Technology), H3K36me3 (4909, Cell Signaling Technology), H3 (4499, Cell Signaling Technology), ACTB (MAB1501, Millipore, clone C4), and GAPDH (sc-26778, Santa Cruz Biotechnology). Densitometry quantification was performed using ImageJ software. All antibodies were used at 1:1,000 dilutions except phos-4EBP1 (1:500), phos-S6K (1:500) and ACTB (1:2,500).

Liu X., Zhang Y., Ni M., Cao H., Signer R.A., Li D., Li M., Gu Z., Hu Z., Dickerson K.E., Weinberg S.E., Chandel N.S., DeBerardinis R.J., Zhou F., Shao Z, & Xu J. (2017). Regulation of Mitochondrial Biogenesis in Erythropoiesis by mTORC1-Mediated Protein Translation. Nature cell biology, 19(6), 626-638.

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Chromatin Profiling of H2Bub1 and H3K4me3

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Chromatin immunoprecipitation was performed as described previously^{35 (link)} 72 h after transfection with control or RNF40 siRNAs using antibodies against H2Bub1 (Cat. No. 5546 S, Cell Signaling Technology) and H3K4me3 (Cat. No. C15410003-50, Diagenode). Next-generation sequencing libraries were prepared using the Microplex Library Preparation kit v2 (Diagenode, Cat.No. C05010011) according to manufacturer’s instructions and samples were sequenced (single-end 50 bp) on a HiSeq4000 (Illumina) at the NGS Integrative Genomics Core Unit (NIG) at the University Medical Center Göttingen (ArrayExpress accession E-MTAB-9234). Processing of sequencing data was performed in the Galaxy environment (galaxy.gwdg.de). Briefly, ChIP-seq reads were mapped to the hg19 reference genome assembly using Bowtie2 (version 2.3.2.2). PCR duplicates were removed using the RmDup tool (version 2.0.1). The deeptools suite (version 3.2.0.0.1) was utilized to generate normalized coverage files (bamCoverage), call peak changes (bigwigCompare), and to generate aggregate plots and heatmaps (computeMatrix and plotHeatmap). Occupancy profiles were visualized using the Integrative Genomics Viewer (IGV 2.4.8). A detailed analysis workflow is available in Supplementary Data.

Wegwitz F., Prokakis E., Pejkovska A., Kosinsky R.L., Glatzel M., Pantel K., Wikman H, & Johnsen S.A. (2020). The histone H2B ubiquitin ligase RNF40 is required for HER2-driven mammary tumorigenesis. Cell Death & Disease, 11(10), 873.

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Comprehensive Antibody Panel for Chromatin Studies

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BAP1 (1/500 dilution; C-4; sc-28383), FOXK1 (1/500 dilution; G-4; sc-373810), YY1 (1/500 dilution; sc-7341) and RAR-alpha (1/500 dilution; sc-551 × ) antibodies were purchased from Santa-Cruz; FLAG (1/1000 dilution; M2; F1804) was purchased from Sigma; HCFC1 (1/1000 dilution; A301-400A) and RNF2 (1/1000 dilution; A302-869A) antibodies were purchased from Bethyl Laboratories; Lamin B1 (1/3000 dilution; ab16048); RING1A (1/1000 dilution; 2820S), RING1B (1/1000 dilution; D22F2; 5694S) H2AK119ub1 (1/3000 dilution; D27C4; 8240S), H3K27me3 (1/3000 dilution; C36B11, 9733S), H3K4me3 (1/3000 dilution; C42D8, 9751) and H4 (1/3000 dilution; 2935S) antibodies, were purchased from Cell Signaling Technology; H2A.Z (1/1000 dilution; 39113), H2B (1/1000 dilution; 5HH2-2A8; 61037); H2BK120ub (1/1000 dilution; C56; 39623), H3 (1/3000 dilution; C-terminal; 39163), and KDM1B (1/1000 dilution; 61457) antibodies were purchased from Active Motif; H3K4me2 (1/3000 dilution; MCA-MAB10003-100-Ex) antibody was purchased from Cosmo Bio; alpha-Tubulin (1/3000 dilution; 1F4E3; A01410) was purchased from Genscript.

Campagne A., Lee M.K., Zielinski D., Michaud A., Le Corre S., Dingli F., Chen H., Shahidian L.Z., Vassilev I., Servant N., Loew D., Pasmant E., Postel-Vinay S., Wassef M, & Margueron R. (2019). BAP1 complex promotes transcription by opposing PRC1-mediated H2A ubiquitylation. Nature Communications, 10, 348.

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Comprehensive Immunohistochemistry Profiling

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The antibodies used were: ASCL1 (Abcam, #ab211327), INSM1 (Abcam, #ab170876), E2F7 (Abcam, ab245655), RB1 (Abcam, ab181616), SYP (Santa Cruz, #sc-17750), RCOR1 (Santa Cruz, #sc-376567), CCN1 (Santa Cruz, #sc-374129), CCN2 (Santa Cruz, #sc-365970), YAP/TAZ (Santa Cruz, #sc-101199), P53 (Santa Cruz, #sc-126), E2F1 (Santa Cruz, #sc-251), GAPDH (Santa Cruz, #sc-47724), LATS1 (Cell signaling, #3477), LATS2 (Cell signaling, #5888), p-LATS (Cell signaling, #8654), H3 (Cell signaling, #4499), H3K27ac (Cell signaling, #8173), H3K4me3 (Cell signaling, #9751), VIN (Cell signaling, #13901), Flag (Sigma, #1804),

Wu Z., Su J., Li F.L., Chen T., Mayner J., Engler A., Ma S., Li Q, & Guan K.L. (2023). YAP silencing by RB1 mutation is essential for small-cell lung cancer metastasis. Nature Communications, 14, 5916.

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