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H3k27me3

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H3K27me3 is a histone modification antibody that recognizes the tri-methylated form of lysine 27 on histone H3. This epigenetic mark is associated with transcriptional repression and heterochromatin formation.

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Lab products found in correlation

Laemmli buffer, by Merck Group (2 mentions) Total h3, by Abcam (2 mentions) Ultra sensitive autoradiography film, by Cytiva (2 mentions) Nupage 4 12 bis tris protein gel, by Thermo Fisher Scientific (2 mentions) Nitrocellulose membrane, by Bio-Rad (2 mentions) Dc protein assay, by Bio-Rad (2 mentions) H3k27ac, by Active Motif (2 mentions) Protein a g magnetic bead, by Thermo Fisher Scientific (1 mentions) H3k27ac, by Thermo Fisher Scientific (1 mentions) Foxa1, by Abcam (1 mentions) H3k27me3, by Merck Group (1 mentions) Hiseq 2500, by Illumina (1 mentions) Zyla 4.2 scmos camera, by Oxford Instruments (1 mentions) Plan apochromat oil immersion objective, by Nikon (1 mentions) Phospho h3s10, by Abcam (1 mentions) H3k9me2, by Cell Signaling Technology (1 mentions) Fibrillarin, by Abcam (1 mentions) Ti e inverted microscope, by Nikon (1 mentions) H3k9ac, by Cell Signaling Technology (1 mentions) Vectastain elite kit, by Vector Laboratories (1 mentions)

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Anti-H3K27me3

6 protocols using h3k27me3

1

Western Blot Analysis of Histone Modifications

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Cells or tumor tissues were lysed in Laemmli buffer (Sigma) and cleared. Protein concentration was determined using DC protein assay (Bio-rad). Proteins (25 μg) were separated on precast NuPAGE 4–12% Bis-Tris protein gels (ThermoFisher), and transferred onto nitrocellulose membranes (Bio-Rad). After blocking with 5% BSA buffer for 1 hour at room temperature, membranes were incubated with primary antibodies overnight at the recommended concentrations. HRP conjugated secondary antibodies (1:10,000; Bio-rad; Cat#5196–2504) were applied, and ultra-sensitive autoradiography film (Amersham) was used to detect the chemiluminescence signal. Primary antibodies used are H3K27me3 (1:1,000, Invitrogen Cat#MA5–11198) and H3 total (1:1,000, Abcam Cat#1791).
Guo H., Vuille J.A., Wittner B.S., Lachtara E.M., Hou Y., Lin M., Zhao T., Raman A.T., Russell H.C., Reeves B.A., Pleskow H.M., Wu C.L., Gnirke A., Meissner A., Efstathiou J.A., Lee R.J., Toner M., Aryee M.J., Lawrence M.S., Miyamoto D.T., Maheswaran S, & Haber D.A. (2023). DNA hypomethylation silences antitumor immune genes in early prostate cancer and CTCs. Cell, 186(13), 2765-2782.e28.
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2

Western Blot Analysis of Histone Modifications

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Cells or tumor tissues were lysed in Laemmli buffer (Sigma) and cleared. Protein concentration was determined using DC protein assay (Bio-rad). Proteins (25 μg) were separated on precast NuPAGE 4–12% Bis-Tris protein gels (ThermoFisher), and transferred onto nitrocellulose membranes (Bio-Rad). After blocking with 5% BSA buffer for 1 hour at room temperature, membranes were incubated with primary antibodies overnight at the recommended concentrations. HRP conjugated secondary antibodies (1:10,000; Bio-rad; Cat#5196–2504) were applied, and ultra-sensitive autoradiography film (Amersham) was used to detect the chemiluminescence signal. Primary antibodies used are H3K27me3 (1:1,000, Invitrogen Cat#MA5–11198) and H3 total (1:1,000, Abcam Cat#1791).
Guo H., Vuille J.A., Wittner B.S., Lachtara E.M., Hou Y., Lin M., Zhao T., Raman A.T., Russell H.C., Reeves B.A., Pleskow H.M., Wu C.L., Gnirke A., Meissner A., Efstathiou J.A., Lee R.J., Toner M., Aryee M.J., Lawrence M.S., Miyamoto D.T., Maheswaran S, & Haber D.A. (2023). DNA hypomethylation silences antitumor immune genes in early prostate cancer and CTCs. Cell, 186(13), 2765-2782.e28.
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3

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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4

Immunofluorescence Imaging of Chromatin and Nuclear Proteins

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Cells were fixed and prepared for indirect immunofluorescence and confocal microscopy as previously described11 ,26 (link).
Images were acquired on a Nikon Ti-E inverted microscope (Nikon) with a Yokogawa CSU-22 spinning disk confocal head with the Borealis modification or a Ti2 inverted microscope fitted with a CSU-W1 spinning disk. Z-stacks of 0.4–0.7 μm spacing were collected using a CoolSnap HQ2 CCD camera (Photometrics) or a Zyla 4.2 sCMOS camera (Andor) with a ×60/1.40 NA or a ×100/1.45 NA Plan Apochromat oil-immersion objective (Nikon).
The following antibodies were used for indirect immunofluorescence imaging: phospho γH2AX (Ser139) (Millipore, 05-636-I; 1:400); H3K27ac (Active Motif, 39133; 1:200); MDC1 (Abcam, ab11171; 1:1,000); MDC1 (Sigma-Aldrich, M2444; 1:1,000); phospho RNA PolII S5 (Millipore, MABE954, clone 1H4B6; 1:400); Cdk9 (Cell Signaling, 2316; 1:10); CDK12 (Abcam, ab246887; 1:400); 53BP1 (Santa Cruz, 22760S; 1:100); H3K27me3 (ThermoFisher Scientific, MA511198; 1:1,000); H3K9ac (Cell Signaling, 9649S; 1:400); H3K9me2 (Cell Signaling, 9753S; 1:400); POM121 (Proteintech, 15645-1-AP; 1:200); phospho H3T3 (Millipore, 07-424, 1:12,000); phospho H3S10 (Abcam, ab47297; 1:200); and fibrillarin (Abcam, ab4566; 1:500). Staining of Dam-methylated DNA in fixed cells was done using purified GFP-tagged m6A-Tracer protein as previously described53 (link).
Papathanasiou S., Mynhier N.A., Liu S., Brunette G., Stokasimov E., Jacob E., Li L., Comenho C., van Steensel B., Buenrostro J.D., Zhang C.Z, & Pellman D. (2023). Heritable transcriptional defects from aberrations of nuclear architecture. Nature, 619(7968), 184-192.
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5

Immunohistochemistry of Epigenetic Markers

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IHC was performed using a Vectastain Elite kit (AK-5001, Vector Laboratories, Burlingame, CA) as described previously34 (link),42 (link),43 (link). Primary antibodies included the human-specific Ki67 (1:50) (Thermo Fisher, Waltham, MA), and rabbit anti-EZH2 (1:50) (Thermo Fisher, Waltham, MA) and H3K27me2 (1:100) (ABCAM INC, Cambridge, CA), and H3K27me3(1:50) (Thermo Fisher, Waltham, MA).
Mackenzie A., Hallam N., Mitchell E, & Beattie T. (1999). Near patient testing for respiratory syncytial virus in paediatric accident and emergency: prospective pilot study. BMJ : British Medical Journal, 319(7205), 289-290.
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6

ChIP Profiling of Histone Modifications

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ChIP was performed on human atrial appendage and mouse cardiac tissue using a Zymo-Spin™ ChIP Kit (Zymo Research, Irvine, CA, Catalog #: D5210), per manufacturer's instructions. Briefly, tissue was homogenized using a PowerGen™ 500 tissue homogenizer (Fisher Scientific, Waltham, MA) and transiently crosslinked with 37% formaldehyde (prepared fresh). Crosslinking was stopped with 1M glycine and chromatin shearing was conducted using Q125 Sonicator (Qsonica, Newtown, CT, Catalog #: Q125-110) for four cycles (30 sec “ON”, 30 sec “OFF”, 40% amplitude) on ice. Chromatin marks studied in the experiment were histone 3 lysine 4 tri-methylation (H3K4me3, Thermo Fisher, Rockford IL, Catalog #: G.532.8) and histone 3 lysine 27 tri-methylation (H3K27me3, Thermo Fisher, Rockford IL, Catalog #: G.299.10). The DNA recovered from the ChIP procedure was then used for qPCR (Supplemental Table 2).
Shepherd D.L., Hathaway Q.A., Nichols C.E., Durr A.J., Pinti M.V., Hughes K.M., Kunovac A., Stine S.M, & Hollander J.M. (2018). Mitochondrial Proteome Disruption in the Diabetic Heart Through Targeted Epigenetic Regulation at the Mitochondrial Heat Shock Protein 70 (mtHsp70) Nuclear Locus. Journal of molecular and cellular cardiology, 119, 104-115.
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