H3k4me3

Manufactured by Santa Cruz Biotechnology

Sourced in United States

H3K4me3 is a histone modification that marks the tri-methylation of lysine 4 on the histone H3 protein. It is a commonly used epigenetic marker that is associated with active gene transcription.

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5 protocols using h3k4me3

Immunodetection of Cellular Proteins

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The antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz), including mouse monoclonal antibodies against Rho A, MMP‐9, AKT1, β‐catenin, N‐cadherin, histone H3 lysine 4 (H3K4me3), proliferating cell‐nuclear antigen (PCNA) and β‐actin. The antibodies were respectively obtained by Abcam Technology (Abcam) and Cell Signalling Technology, including mouse/rabbit polyclonal antibodies against RhoA Ser188, AKT Thr180Tyr182 and NFκB p50 bought from all culture materials were obtained from Gibco (Grand Island, NY, USA). Protease inhibitor cocktails, reactive oxygen species (ROS) scavenger (N‐acetyl cysteine [NAC]), dihydroethidium (DHE), NFκB inhibitor (PDTC), SDS, RhoGTPase inhibitor (CCG‐1423), NP‐40, phosphoinositide 3‐kinase inhibitor (wortmannin), sodium deoxycholate, 2,7‐dichlorodihydrofluorescein diacetate (H2DCFDA) and 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) were purchased from Sigma. Rabbit polyclonal antibodies against CHGA and UCHL1 were purchased from Bioss Inc.

Lee K., Chen H., Cheng K., Liu T., Lee K., Teng C., Huang C., Hsieh M, & Kuo H. (2023). Use of iTRAQ‐based quantitative proteomic identification of CHGA and UCHL1 correlated with lymph node metastasis in colorectal carcinoma. Journal of Cellular and Molecular Medicine, 27(14), 2004-2020.

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ChIP Analysis of Histone Modifications

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ChIP was performed using 3 μl per sample of the following antibodies: MTF2 (Aviva System Biology ARP34292, lot QC49692-42166), H3K27me3 (Millipore 07-449, lot 2717675), EZH2 (Diagenode C15410039, lot 003), H3K4me3 (Ab858, lot GR240214-4), and Anti-GATA-2 Antibody (H-6) (Santa Cruz, #sc-515178, 1:500).

Loh C.H., van Genesen S., Perino M., Bark M.R, & Veenstra G.J. (2021). Loss of PRC2 subunits primes lineage choice during exit of pluripotency. Nature Communications, 12, 6985.

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Antibodies Used for Chromatin Analysis

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The antibodies used in this study were purchased from the following sources: Millipore (Billerica, MA): ASYM25 (09-814), HP1 (MAB3448), histone H3 (06-755), H3ac (06-599), H3K4me3 (07-473), H3K9me2 (07-441), H3K9me3 (07-442), H3K27me3 (07-449), H4 (05-858), H4ac (06-598), Prmt1 (07-404) and Sox2 (AB5603); Santa Cruz Biotech (Santa Cruz, CA): actin (sc-47778), c-Myc (sc-40, sc-789), GAPDH (sc-166574), Gata4 (sc-9053), Gata6 (sc-9055), GST (sc-138), mSin3a (sc-994), Oct4 (sc-5279) and p300 (sc-32244); Cell Signaling Technology (Danvers, MA): HDAC1 (2062), HDAC2 (2545s) and KLF4 (4038); Abcam (Cambridge, UK): H3R17me2 (ab412); Sigma (St. Louis, MO): FLAG (F3165); MBL (Japan): FLAG (PM020); Active Motif (Carlsbad, CA): H4R3me2a (39705); R&D Systems (Minneapolis, MN): KLF4 (AF3158); and Bethyl Laboratories (Montgomery, TX): Nanog (A300-397A).
A specific monoclonal antibody against Klf4-R396me2a was generated by Absea Biotechnology Ltd (Beijing, China) using synthesized peptide CGRRSWPRKRTATHT, corresponding to residues aa 387–402 of Klf4, as an antigen.

Zuo Z.Y., Yang G.H., Wang H.Y., Liu S.Y., Zhang Y.J., Cai Y., Chen F., Dai H., Xiao Y., Cheng M.B., Huang Y, & Zhang Y. (2022). Klf4 methylated by Prmt1 restrains the commitment of primitive endoderm. Nucleic Acids Research, 50(4), 2005-2018.

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Chromatin Immunoprecipitation for Stem Cell Differentiation

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Single ChIP-seq experiment for each H3K4me3, H3K27ac, H3K27ac, and H3K9me3 during differentiation of TSCs into STs and EVTs has been performed following the established procedures^{57 (link)}. In brief, TSCs were fixed with 1% formaldehyde for 7 min at room temperature and then quenched with glycine for 5 min. The fixed cells were sonicated using a Bioruptor (Diagenode) with a 30-s on and 1-min off setting, repeated for 10 min (three cycles). These sheared chromatins were used for immunoprecipitation with 10 µg of native antibodies, including H3K4me3 (Santa Cruz, sc-585), H3K27ac (Santa Cruz, sc-7202X), H3K27me3 (Santa Cruz, sc-9008X), and H3K9me3 (Santa Cruz, sc-8977). The enriched ChIP materials were utilized to generate next-generation sequencing libraries with the NEB ChIP-seq library preparation kit (NEB, E7370L). Subsequently, the ChIP-seq libraries were sequenced using the Illumina NextSeq 500 machine from Illumina.

Lee B.K., Salamah J., Cheeran E, & Adu-Gyamfi E.A. (2024). Dynamic and distinct histone modifications facilitate human trophoblast lineage differentiation. Scientific Reports, 14, 4505.

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ChIP-qPCR analysis of ABCB1 promoter

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D283 and HD-MB03 cells were cross-linked in 1% paraformaldehyde for 10 min. Cell lysis, shearing, immunoprecipitation and DNA purification were undertaken using a Magna ChIP A/G immunoprecipitation kit (Sigma Aldrich (St. Louis, MO, USA); 17-10085) according to the manufacturer’s instructions. Samples were sonicated using a water bath sonicator (Diagenode, Denville, NJ, USA) to obtain 200–500 bp chromatin fragments. The following antibodies were utilised to pull down protein-DNA complexes, with Immunoglobin (IgG) 1 used as a negative control and histone H3K4Me3 used to confirm active promoter regions: YB-1 (Santa Cruz (Santa Cruz, CA, USA); SC101198); IgG1 (Cell Signalling; 5415); H3K4Me3 (Active Motif (Carlsbad, CA, USA); 61379). To quantify target protein interaction with the gene of interest, qPCR using primers specific to an inverted CCAAT box within the ABCB1 promoter region was undertaken. Primer sequences were as follows: ABCB1 forward (5′ CAT GCT GAA GAA AGA CCA CTG C 3′) and ABCB1 reverse (5′ AGG CTT CCT GTG GCA AAG AG 3′).

Taylor L., Wade P.K., Johnson J.E., Aldighieri M., Morlando S., Di Leva G., Kerr I.D, & Coyle B. (2023). Drug Resistance in Medulloblastoma Is Driven by YB-1, ABCB1 and a Seven-Gene Drug Signature. Cancers, 15(4), 1086.

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