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H3k27me3

Manufactured by Merck Group
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H3K27me3 is a lab equipment product that detects and measures the trimethylation of lysine 27 on histone H3 protein. This modification is associated with gene silencing and is involved in various cellular processes. The product provides a reliable and specific method for researchers to study epigenetic regulation and chromatin dynamics.

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Lab products found in correlation

H3k4me3, by Merck Group (66 mentions) H3k9me3, by Abcam (30 mentions) H3k27ac, by Abcam (24 mentions) H3k9me3, by Merck Group (21 mentions) H3k4me3, by Abcam (15 mentions) H3k9me2, by Merck Group (15 mentions) β actin, by Merck Group (13 mentions) H3k9ac, by Merck Group (13 mentions) H3k27ac, by Active Motif (12 mentions) H3k36me3, by Abcam (12 mentions) H3k27ac, by Merck Group (11 mentions) H3k4me2, by Merck Group (11 mentions) H3k4me3, by Active Motif (11 mentions) H3k4me1, by Abcam (9 mentions) Flag tag (flag), by Merck Group (8 mentions) H3k9me3, by Active Motif (8 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (8 mentions) H3k27me2, by Merck Group (7 mentions) H2ak119ub, by Cell Signaling Technology (7 mentions) Bioruptor, by Diagenode (7 mentions)

283 protocols using h3k27me3

1

Western Blot Analysis of Protein Lysates

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Protein lysates were prepared in radioimmunoprecipitation assay buffer and quantified with the Bradford assay (Bio-Rad). Electrophoresis was performed in precast, NuPage 4 to 12% bis-tris protein gels before transferring into polyvinylidene difluoride membranes using the Trans-Blot Turbo transfer system (Bio-Rad). Before blocking and antibody incubation, membranes were stained with Ponceau S reagent (Sigma-Aldrich, #P3504) to visualize protein bands and cut at specific sizes so different antibodies could be tested at the same time. Western blots were performed using antibodies against H3K27me3 (0.4 μg/ml; Sigma-Aldrich, rabbit polyclonal), Isl1 (1:10,000; rabbit monoclonal, Abcam), Hsp90 (1:2000; rabbit polyclonal, Cell Signaling Technology), Gapdh (1:2000; rabbit monoclonal, Cell Signaling Technology), a goat anti-rabbit immunoglobulin G, and horseradish peroxidase–linked antibody (1:2000; Cell Signaling Technology). Densitometry analysis of Western blots was performed using Fiji ImageJ.
Hatzistergos K.E., Durante M.A., Valasaki K., Wanschel A.C., Harbour J.W, & Hare J.M. (2020). A novel cardiomyogenic role for Isl1+ neural crest cells in the inflow tract. Science Advances, 6(49), eaba9950.
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2

ChIP-seq Analysis of Histone Modifications

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The ChIP-seq assay was performed according to a published protocol [31 (link)]. Two commercial antibodies, H3K27me3 (Sigma, Cat. #07–449) and H3K27ac (Sigma, Cat. #07–360), were used for immunoprecipitation. The ChIP DNAs were ligated with Illumina sequencing adaptors and sequenced using the Illumina HiSeq platform to produce 150 bp paired-end reads. The raw reads generated from ChIP-seq were quality filtered and trimmed using trim_galore. Cleaned reads were mapped to the B. distachyon genome using Bowtie2 v.2.2.5 [50 (link)] with default parameters. Mapped reads were filtered using SAMtools v.1.9 [51 (link)] to retain only correctly read pairs with a mapping quality score ≥ 10 for further analysis. Peak calling was performed using MACS2 v.2.1.4 [52 (link)] with the parameters “-f BAMPE –broad -g 2.7e8 –nomodel”.
Zhang X., Yu G., Dai Y., Zhang H., Wang K, & Han J. (2023). High-resolution Hi-C maps highlight multiscale chromatin architecture reorganization during cold stress in Brachypodium distachyon. BMC Plant Biology, 23, 260.
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3

Antibody Characterization for Protein Analysis

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Antibodies used were as follows: EZH2 (Cell Signaling, #5246, Danvers, MA, USA), H3K27me3 (Active motif, #61017, Carlsbad, CA, USA), Beta Actin (Sigma, #AC-74), Histone 3 (AbCam, Cambridge, UK, ab1791), AIF (Santa Cruz, #sc-5586, Dallas, Texas USA), NDRG1_pT346 (Cell Signaling #3217), p53 (Santa Cruz, #sc-126), Noxa (Imgenex, Littleton, CO, USA, #114C307.1), Mcl-1 (BD Biosciences, #559027). Additional antibodies are listed in Supplementary Table S2.
Tiffen J.C., Gunatilake D., Gallagher S.J., Gowrishankar K., Heinemann A., Cullinane C., Dutton-Regester K., Pupo G.M., Strbenac D., Yang J.Y., Madore J., Mann G.J., Hayward N.K., McArthur G.A., Filipp F.V, & Hersey P. (2015). Targeting activating mutations of EZH2 leads to potent cell growth inhibition in human melanoma by derepression of tumor suppressor genes. Oncotarget, 6(29), 27023-27036.
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4

Protein Extraction and Western Blotting

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For worm samples, proteins were extracted as described previously13 (link). Western blotting was performed with antibodies against GFP (1:1000, Cat. 2956, CST), Actin (1:2000, Cat. A5441, Sigma), H3K18Ac (1:1000, Cat. 07-354, Merck), H3K27Ac (1:1000, Cat. ab4729, abcam), H3K9Ac (1:1000, Cat. 06-942, Merck), H3K4Ac (1:1000, Cat. Ab176799, abcam), Histone 3 (1:1000, Cat. 9715, CST), Tubulin (1:2000, Cat. T5168, Sigma), H3K27Me3 (1:1000, Cat. 07-449, millipore), H3K27Me2 (1:1000, Cat. ab24684, abcam), H3K27Me1 (1:1000, Cat. 07-448, millipore), H3K9Me1 (1:1000, Cat. 07-450, millipore), H3K4Me3 (1:1000, Cat. 07-473, millipore), Histone 4 (1:1000, Cat. sc-10810, Santa Cruz), H4K5Ac (1:1000, Cat. ab51997, abcam), β-amyloid 1–16 (6E10) (1:1000, Cat. 803001, BioLegend), HA-tag (1:2000, Cat. 3724, CST), Flag-tag (1:1000, Cat. F7425, Sigma), Myc-tag (1:2000, Cat. sc-40, Santa Cruz), GST-tag (1:1000, Cat. 2625, CST), AcK (1:1000, Cat. 9441, CST), AcK (1:1000, Cat. 9814, CST) and HRP-labelled anti-rabbit (Cat. 7074, CST) and anti-mouse (Cat. 7076, CST) secondary antibodies.
Li T.Y., Sleiman M.B., Li H., Gao A.W., Mottis A., Bachmann A.M., El Alam G., Li X., Goeminne L.J., Schoonjans K, & Auwerx J. (2021). The transcriptional coactivator CBP/p300 is an evolutionarily conserved node that promotes longevity in response to mitochondrial stress. Nature aging, 1(2), 165-178.
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5

Chromatin Immunoprecipitation of SMC Contractile Genes

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Chromatin Immunoprecipitation (ChIP) was performed as previously described (Dahl and Collas, 2008 (link); Gomez et al., 2013 (link)). In brief, passage-matched control, Myocd-LSD1, Myocd-LSD1NF SMC were fixed with 1% PFA for 10 min at room temperature. Cells were sonicated with a Bioruptor Pico (Diagenode) to obtain chromatin fragments of 200-500 base pairs. Chromatin was incubated with Protein G Dynabeads (Invitrogen, 10004D) and one of the following antibodies: H3K4me2 (2μg; #07-030, Millipore), Flag (4;g #F3165, Sigma Aldrich), H3K9me3 (2μg; ab176916, Abcam), H3K27me3 (2μg; 07-449, Sigma Aldrich), SRF (2μg; sc-13029, Santa Cruz), KLF4 (2μg; sc-20691, Santa Cruz), TET2 (5μg; ABE364, Millipore), H3ac (2μg; 06-599, Millipore), rabbit IgG (Abcam, ab171870) or mouse IgG (Abcam, ab37355). Genomic DNA was extracted with phenol-chloroform from immunoprecipitated (IP) and non-immunoprecipitated (INPUT) samples. Histone modification and protein enrichment was measured by qPCR using primer sets targeting CArG regions of the SMC contractile genes. Results were expressed as IP/INPUT. Primers used for ChIP-qPCR are listed in Key Resources Table.
Liu M., Espinosa-Diez C., Mahan S., Du M., Nguyen A.T., Hahn S., Chakraborty R., Straub A.C., Martin K.A., Owens G.K, & Gomez D. (2021). H3K4 di-methylation governs smooth muscle lineage identity and promotes vascular homeostasis by restraining plasticity. Developmental cell, 56(19), 2765-2782.e10.
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6

Epigenetic Regulation of Gene Expression

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DMEM-F12 was bought from GIBCO (USA). The charcoal-stripped fetal calf serum (FCS) was purchased from HyClone Laboratories (Inc., Logan, UT, USA). Anti-mouse Flag-tag, HA-tag, H3, H3K27me3 and GAPDH were bought from Sigma-Aldrich (Inc, USA). Cell counting kit-8 was bought from Dojindo Laboratories (Inc, Japan). The EZH2 inhibitor EPZ6438 and BET inhibitor JQ-1 were from Selleck Chemicals.
Zhang Y., Dong W., Zhu J., Wang L., Wu X, & Shan H. (2017). Combination of EZH2 inhibitor and BET inhibitor for treatment of diffuse intrinsic pontine glioma. Cell & Bioscience, 7, 56.
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7

Immunoblotting Analysis of Stem Cell Markers

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Western blots were performed by extracting cell pellets using RIPA buffer. Protein concentrations were quantified using Bradford Assay [37 (link)]. Antibodies were used according to the manufacturer’s instructions: BCOR (12107-1-AP) (from Proteintech, Rosemont, IL USA), GFAP (#3670), Histone H3 (#14269), H2AK119Ub (#8240), H2A (#12349), Nestin (#33475) (from Cell Signaling Technologies, Beverly, MA, USA), β-Actin (#47778), MAP2 (#20172) (from Santa Cruz Biotechnology), Tuj1 (MAB5564), H3K27me3 (07-449), Olig2 (AB9610) (from Sigma-Aldrich, St. Louis, MO, USA), H3K36me2 (#39255) (from Active Motif, Carlsbad, CA, USA), SOX2 (AM2048a) (from Abcepta, San Diego, CA, USA). Densitometry was performed using ImageJ v1.440 software.
Nakata S., Yuan M., Rubens J.A., Kahlert U.D., Maciaczyk J., Raabe E.H, & Eberhart C.G. (2021). BCOR Internal Tandem Duplication Expression in Neural Stem Cells Promotes Growth, Invasion, and Expression of PRC2 Targets. International Journal of Molecular Sciences, 22(8), 3913.
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8

Protein Extraction and Western Blotting

Cited 1 time
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For worm samples, proteins were extracted as described previously13 (link). Western blotting was performed with antibodies against GFP (1:1000, Cat. 2956, CST), Actin (1:2000, Cat. A5441, Sigma), H3K18Ac (1:1000, Cat. 07-354, Merck), H3K27Ac (1:1000, Cat. ab4729, abcam), H3K9Ac (1:1000, Cat. 06-942, Merck), H3K4Ac (1:1000, Cat. Ab176799, abcam), Histone 3 (1:1000, Cat. 9715, CST), Tubulin (1:2000, Cat. T5168, Sigma), H3K27Me3 (1:1000, Cat. 07-449, millipore), H3K27Me2 (1:1000, Cat. ab24684, abcam), H3K27Me1 (1:1000, Cat. 07-448, millipore), H3K9Me1 (1:1000, Cat. 07-450, millipore), H3K4Me3 (1:1000, Cat. 07-473, millipore), Histone 4 (1:1000, Cat. sc-10810, Santa Cruz), H4K5Ac (1:1000, Cat. ab51997, abcam), β-amyloid 1–16 (6E10) (1:1000, Cat. 803001, BioLegend), HA-tag (1:2000, Cat. 3724, CST), Flag-tag (1:1000, Cat. F7425, Sigma), Myc-tag (1:2000, Cat. sc-40, Santa Cruz), GST-tag (1:1000, Cat. 2625, CST), AcK (1:1000, Cat. 9441, CST), AcK (1:1000, Cat. 9814, CST) and HRP-labelled anti-rabbit (Cat. 7074, CST) and anti-mouse (Cat. 7076, CST) secondary antibodies.
Li T.Y., Sleiman M.B., Li H., Gao A.W., Mottis A., Bachmann A.M., El Alam G., Li X., Goeminne L.J., Schoonjans K, & Auwerx J. (2021). The transcriptional coactivator CBP/p300 is an evolutionarily conserved node that promotes longevity in response to mitochondrial stress. Nature aging, 1(2), 165-178.
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9

Transcription Factor and Histone Modification ChIP-seq

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ChIP‐seq for AR, FOXA1, CTCF, H3K27ac, and H3K27me3 were performed using 5 μg of antibody and 50 μL of Protein A/G magnetic beads (Invitrogen, Carlsbad, CA, USA) per sample. Antibodies applied here were AR (Millipore, Burlington, MA, USA, 06‐680, lot#2489005&2943813); FOXA1 (Abcam, Cambridge, UK, ab5089, lot#GR20766‐16); CTCF (Millipore, 07‐729, lot#2887267); H3K27ac (Active Motif, Carlsbad, CA, USA 39133, lot#31814008); H3K27me3 (Sigma‐Aldrich, St. Louis, MO, USA 07‐449, lot#2826067). As control, input of each sample was generated separately. Immunoprecipitated DNA was processed for sequencing using standard protocols and sequenced on an Illumina Hi‐seq 2500 with 65 bp single end reads.
Severson T.M., Zhu Y., De Marzo A.M., Jones T., Simons J.W., Nelson W.G., Yegnasubramanian S., Freedman M.L., Wessels L., Bergman A.M., Haffner M.C, & Zwart W. (2021). Epigenetic and transcriptional analysis reveals a core transcriptional program conserved in clonal prostate cancer metastases. Molecular Oncology, 15(7), 1942-1955.
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10

Quantifying H3K27me3 Levels in RGCs

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To analyze H3K27me3 levels in RGCs, retinas were dissected from transcardially perfused mice 2 weeks after intravitreal injection of AAV2-GFP, AAV2-Ezh2, or AAV2-Ezh2-Y726D and sectioned with a cryostat. Retinal sections were stained with guinea pig anti-Rbpms (1:500, Sigma-Aldrich ABN1376) and mouse anti-H3K27me3 (1:100, Sigma-Aldrich 05-1951) following the steps described above (see Immunofluorescence of retinal sections).
To quantify H3K27me3 levels in RGCs, fluorescence intensity of H3K27me3 immunoreactivity of at least 150 Rbpms-positive cells from 10–12 nonadjacent retinal sections acquired with identical imaging configurations was analyzed for each retina. Fluorescence intensity was measured using the “outline spline” function of the AxioVision software and the background fluorescence intensity was subtracted.
Wang X.W., Yang S.G., Hu M.W., Wang R.Y., Zhang C., Kosanam A.R., Ochuba A.J., Jiang J.J., Luo X., Guan Y., Qian J., Liu C.M, & Zhou F.Q. (2024). Histone methyltransferase Ezh2 coordinates mammalian axon regeneration via regulation of key regenerative pathways. The Journal of Clinical Investigation, 134(3), e163145.
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