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H3k27me3 c36b11

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H3K27me3 (C36B11) is a monoclonal antibody that recognizes the trimethylated form of histone H3 at lysine 27. This antibody is commonly used in chromatin immunoprecipitation (ChIP) and immunoblotting applications to study the epigenetic regulation of gene expression.

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Lab products found in correlation

Ezh2 d2c9, by Cell Signaling Technology (3 mentions) Ab76291, by Cell Signaling Technology (2 mentions) Epr17341, by Abcam (2 mentions) Antibodies specific for total histone h3 96c10, by Cell Signaling Technology (2 mentions) H3k27me2 d18c8, by Cell Signaling Technology (2 mentions) Histone h3.3 antibody ab97968, by Abcam (2 mentions) Histone k27m mutant antibody abe419, by Merck Group (2 mentions) Enhanced chemi luminescence (ecl), by GE Healthcare (2 mentions) H3k27me1, by Cell Signaling Technology (2 mentions) Ac 74, by Merck Group (2 mentions) Nupage transfer buffer, by Thermo Fisher Scientific (2 mentions) Nupage bis tris polyacrylamide gel, by Thermo Fisher Scientific (2 mentions) Supersignal west pico, by Thermo Fisher Scientific (2 mentions) Lds loading buffer, by Thermo Fisher Scientific (2 mentions) H3k4me2 c75h12, by Cell Signaling Technology (2 mentions) H3k36me2 c64g9, by Cell Signaling Technology (2 mentions) Supersignal west femto chemiluminescence kit, by Thermo Fisher Scientific (2 mentions) Supersignal western blot enhancer kit, by Thermo Fisher Scientific (2 mentions) 0.2 μm pvdf membrane, by Bio-Rad (2 mentions) H3k4me3, by Cell Signaling Technology (2 mentions)

12 protocols using h3k27me3 c36b11

1

Histone Modification and DNA Replication Assays

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Histone H3 (WB 1:5000), H3K9me1 (WB 1:1000), H3K9me3 (WB 1:1000) and Mcm3 (WB 1:2000) were generated in the laboratory and were used previously (25 (link),26 (link)). Anti-GFP (ab6556, WB 1:10 000), Polδ (ab186407, WB 1:1000), Polϵ (ab74308, WB 1:1000), KDM4D (ab93694, WB 1:2000, IF 1:200, ChIP 4 μg per IP), Mcm6 (ab201683, ChIP 4 μg per IP), Orc2 (ab68348, WB 1:2000), anti-SLD5 (ab139683, WB 1:1000) and anti-CldU (ab6326) were purchased from Abcam; Orc5 (sc-20635, WB 1:1000), Cdc45 (sc-20685, WB 1:1000), PCNA (sc-56, WB 1:1000) and Tubulin (sc-9104, WB 1:5000) were purchased from Santa Cruz Biotechnology; H3K27me3 (C36B11, WB 1:1000) and H3S10ph (D2C8, WB 1:1000) were purchased from Cell Signaling Technology.
Fluorescein isothiocyanate (FITC)-conjugated anti-BrdU (347583) and anti-IdU (347580) antibodies were purchased from BD Biosciences; FITC goat anti-mouse, FITC goat anti-rabbit and Cy3 goat anti-rat antibodies were purchased from Jackson ImmunoResearch.
Wu R., Wang Z., Zhang H., Gan H, & Zhang Z. (2016). H3K9me3 demethylase Kdm4d facilitates the formation of pre-initiative complex and regulates DNA replication. Nucleic Acids Research, 45(1), 169-180.
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2

Immunohistochemistry for Trk and BCOR Proteins

Cited 2 times
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Immunohistochemical staining was performed using a pan-Trk antibody (EPR17341; Abcam, Cambridge, MA)[14 (link)], which recognizes Trk proteins including Trk-A, Trk-B, and Trk-C, encoded by the NTRK1, NTRK2, and NTRK3 genes, respectively. Immunostaining for BCOR using clone C-10 (sc-514576; Santa Cruz, Dallas, TX), was performed and/or reviewed, as previously described.[11 (link)] In a small subset of cases additional immunostains were applied including NTRK1 (Ab76291, 1:1,500, ABCAM), H3K27me3 (C36B11 (1:200 dilution; Cell Signaling Technology, Danvers, MA) and TLE1 (Santa Cruz Biotech, clone Poly; sc-9121; 1:100 dilution) .
The staining patterns of pan-Trk were recorded as cytoplasmic, membranous, and/or nuclear. The intensities were recorded as weak, moderate, or strong, and the percentage of tumor cells positive for pan-Trk staining was also assessed in each case. Cases with >5% tumor cells staining for pan-Trk were considered positive. The staining results of BCOR were also recorded whenever available.
Kao Y.C., Sung Y.S., Argani P., Swanson D., Alaggio R., Tap W., Wexler L., Dickson B.C, & Antonescu C.R. (2020). NTRK3 Overexpression in Undifferentiated Sarcomas with YWHAE and BCOR Genetic Alterations. Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc, 33(7), 1341-1349.
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3

CRISPR-Mediated Genetic Manipulation of MPNST Cells

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Published murine MPNST cells (see refs. 30 (link), 32 (link), 34 (link)) were transfected with the px459 plasmid (Addgene 62988) expressing Cas9 and a single guide RNA against Suz12 (ΔSuz12), Eed (ΔEed), or a nontargeting control (tdTomato). (55 (link)) Cells were grown in DMEM (Thermo Fisher Scientific, 11965092) supplemented with 10% fetal bovine serum, 1% sodium pyruvate (Gibco, 11360-070), and 1% penicillin/streptomycin (pen/strep) (Gibco, 15140-122). Clonal cell lines were isolated by limiting dilution, and Suz12 and Eed indels were verified by sequencing followed by ICE analysis (Synthego, https://ice.synthego.com/#/). Western blot confirmed loss of EED (Cell Signaling Technology, EED 51673S), SUZ12 (Cell Signaling Technology, SUZ12 D39F6, 3737S), and H3K27me3 (Cell Signaling Technology, H3K27me3 C36B11, 9733S) in PRC2-deleted cell lines. For cell proliferation assays, 100 μL of resuspended cells (3.367 × 105 cells/mL) were plated into 96 wells in triplicate. Cell number was determined 1, 3, 4, and 5 days postseeding by adding 100 μL of fresh media and 20 μL of alamar blue (MilliporeSigma, R7017) for 2 hours and reading fluorescence at 590 nm (BioTek Synergy HT).
Brockman Q.R., Scherer A., McGivney G.R., Gutierrez W.R., Voigt A.P., Isaacson A.L., Laverty E.A., Roughton G., Knepper-Adrian V., Darbro B., Tanas M.R., Stipp C.S, & Dodd R.D. (2022). PRC2 loss drives MPNST metastasis and matrix remodeling. JCI Insight, 7(20), e157502.
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4

Immunohistochemistry for Trk and BCOR Proteins

Cited 2 times
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Immunohistochemical staining was performed using a pan-Trk antibody (EPR17341; Abcam, Cambridge, MA)[14 (link)], which recognizes Trk proteins including Trk-A, Trk-B, and Trk-C, encoded by the NTRK1, NTRK2, and NTRK3 genes, respectively. Immunostaining for BCOR using clone C-10 (sc-514576; Santa Cruz, Dallas, TX), was performed and/or reviewed, as previously described.[11 (link)] In a small subset of cases additional immunostains were applied including NTRK1 (Ab76291, 1:1,500, ABCAM), H3K27me3 (C36B11 (1:200 dilution; Cell Signaling Technology, Danvers, MA) and TLE1 (Santa Cruz Biotech, clone Poly; sc-9121; 1:100 dilution) .
The staining patterns of pan-Trk were recorded as cytoplasmic, membranous, and/or nuclear. The intensities were recorded as weak, moderate, or strong, and the percentage of tumor cells positive for pan-Trk staining was also assessed in each case. Cases with >5% tumor cells staining for pan-Trk were considered positive. The staining results of BCOR were also recorded whenever available.
Kao Y.C., Sung Y.S., Argani P., Swanson D., Alaggio R., Tap W., Wexler L., Dickson B.C, & Antonescu C.R. (2020). NTRK3 Overexpression in Undifferentiated Sarcomas with YWHAE and BCOR Genetic Alterations. Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc, 33(7), 1341-1349.
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5

Quantitative Analysis of H3K27me3 and EZH2

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H3K27me3 (C36B11) and EZH2 (D2C9) antibodies were purchased from Cell Signaling Technology. Tumor tissues harvested from mice were frozen in OCT. Frozen tumor tissue was fixed in ice-cold methanol and blocked in 5% goat serum in PBS after permeabilization with 0.5%Triton-X. They were then stained at 4°C overnight with primary antibody. Subsequently tissues were washed and stained with secondary antibody goat anti-rabbit IgG (H+L), Alexa flour 488 (Thermo Fisher Scientific; A1108) at room temperature for 1h. Finally, the tissues were stained with 1x DAPI (Molecular Probes/Invitrogen; D3571) washed and mounted using fluorescent mounting agent (DAKO; S3023). For cellular staining, 8-chamber slides were used and coated with 0.1mg/mL Poly-L-lysine. 1x105 cells were cultured for 24-48h and then treated with GSK126 and/or IFNγ for 24h. Cell were then washed in PBS and fixed using 4% paraformaldehyde. Permeabilization, blocking and staining with primary and secondary antibodies were carried out as described above. Once stained the cells were mounted with Vectasheild with DAPI (Vector Laboratories; H1200). All stained tissues and cells were imaged on a Zeiss LSM 880 or Zeiss LSM 780 confocal microscopes. The images were analyzed and quantified using ImageJ software.
Ratnam N.M., Sonnemann H.M., Frederico S.C., Chen H., Hutchinson M.K., Dowdy T., Reid C.M., Jung J., Zhang W., Song H., Zhang M., Davis D., Larion M., Giles A.J, & Gilbert M.R. (2021). Reversing Epigenetic Gene Silencing to Overcome Immune Evasion in CNS Malignancies. Frontiers in Oncology, 11, 719091.
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6

Histone Modification Detection Assay

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Nuclear proteins were extracted from proliferating cells in acid extraction buffer (0.2N HCL), following the extraction of cytoplasmic proteins with TNE buffer: 150 mM NaCl, 10 mM Tris pH 7.4, 1% Triton X-100, 5 mM EDTA, 1% NP40, 1 μM DTT, and proteinase (Roche) plus phosphatase (Sigma) inhibitor cocktails. Protein extracts were resolved by SDS-PAGE and transferred to poly(vinylidene difluoride) (PVDF) membranes. After probing with primary antibodies, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody, and signals visualized by ECL (GE Healthcare). Antibodies specific for total histone H3 (96C10, 1:1,000), H3K27me3 (C36B11, 1:1,000), H3K27me2 (D18C8, 1:1,000), H3K27me1 (#7693 1:1,000), and EZH2 (D2C9, 1:1,000), were obtained from Cell Signaling Technologies. Histone H3.3 antibody (ab97968, 1:1,000) was obtained from Abcam, and histone K27M mutant antibody (ABE419, 1:1,000) was from EMD Millipore.
Hashizume R., Andor N., Ihara Y., Lerner R., Gan H., Chen X., Fang D., Huang X., Tom M.W., Ngo V., Solomon D., Mueller S., Paris P.L., Zhang Z., Petritsch C., Gupta N., Waldman T.A, & James C.D. (2014). Pharmacologic inhibition of histone demethylation as a therapy for pediatric brainstem glioma. Nature medicine, 20(12), 1394-1396.
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7

Histone Modification Detection Assay

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Nuclear proteins were extracted from proliferating cells in acid extraction buffer (0.2N HCL), following the extraction of cytoplasmic proteins with TNE buffer: 150 mM NaCl, 10 mM Tris pH 7.4, 1% Triton X-100, 5 mM EDTA, 1% NP40, 1 μM DTT, and proteinase (Roche) plus phosphatase (Sigma) inhibitor cocktails. Protein extracts were resolved by SDS-PAGE and transferred to poly(vinylidene difluoride) (PVDF) membranes. After probing with primary antibodies, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody, and signals visualized by ECL (GE Healthcare). Antibodies specific for total histone H3 (96C10, 1:1,000), H3K27me3 (C36B11, 1:1,000), H3K27me2 (D18C8, 1:1,000), H3K27me1 (#7693 1:1,000), and EZH2 (D2C9, 1:1,000), were obtained from Cell Signaling Technologies. Histone H3.3 antibody (ab97968, 1:1,000) was obtained from Abcam, and histone K27M mutant antibody (ABE419, 1:1,000) was from EMD Millipore.
Hashizume R., Andor N., Ihara Y., Lerner R., Gan H., Chen X., Fang D., Huang X., Tom M.W., Ngo V., Solomon D., Mueller S., Paris P.L., Zhang Z., Petritsch C., Gupta N., Waldman T.A, & James C.D. (2014). Pharmacologic inhibition of histone demethylation as a therapy for pediatric brainstem glioma. Nature medicine, 20(12), 1394-1396.
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8

Comprehensive Antibody Characterization for Cellular Analysis

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The following primary antibodies (antigen [clone], dilution, vendor, cat. no.) were used for these studies: NPC1 [EPR5209], 1:500, Abcam, ab134113; NeuN [A60], 1:500, MilliporeSigma, MAB377; Ki67 [polyclonal], 1:200, Abcam, ab15580; Vinculin [hVIN-1], 1:2000, MilliporeSigma, V9131; Neurofilament [N52], 1:500, MilliporeSigma, MAB5266; H3K27me3 [C36B11], 1:200 [IF], 1:1000 [WB], Cell Signaling Technologies, 9733 S; H3K9me3 [D4W1U], 1:200 [IF], 1:1000 [WB], Cell Signaling Technologies, 13969 S; Histone H3 [96C10], 1:2000, Cell Signaling Technologies, 3638 S; MBP12 (link), 1:100 [IF], 1:500 [WB], Abcam, ab7349; OLIG2 [polyclonal], 1:500 [IF], 1:1000 [WB], MilliporeSigma, AB9610; SOX10 [SP267], 1:400, Abcam, ab227680; SOX10 [A-2], 1:200, Santa Cruz Biotechnology, sc-365692; H3K27me3 [polyclonal], ActiveMotif, cat. #39155; H3K27ac [polyclonal], ActiveMotif, cat. #39133.
Kunkel T.J., Townsend A., Sullivan K.A., Merlet J., Schuchman E.H., Jacobson D.A, & Lieberman A.P. (2023). The cholesterol transporter NPC1 is essential for epigenetic regulation and maturation of oligodendrocyte lineage cells. Nature Communications, 14, 3964.
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9

Quantifying Histone Modifications by Western Blot

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Equal numbers of cells from each population were sorted into trichloroacetic acid (TCA, Sigma), and the TCA final concentration was adjusted to 10%. Samples were centrifuged at 16,000g for 15 minutes, and precipitates were washed twice with cold acetone and dried. Samples were solubilized in 9M urea, 2% Triton X-100, 1% DTT. LDS loading buffer (Life Technologies) was added. Samples were heated at 70°C for 10 minutes, separated on NuPAGE Bis-Tris polyacrylamide gels (Life Technologies), and transferred to 0.2μm PVDF membranes (BioRad) by wet transfer using NuPAGE transfer buffer (Life Technologies). Western blots were performed using antibodies against β-actin (AC-74, Sigma), histone H3 (polyclonal, Abcam, ab1791), H3K4me3 (C42D8, Cell Signaling), H3K27me3 (C36B11, Cell Signaling), H3K4me2 (C75H12, Cell Signaling) and H3K36me2 (C64G9, Cell Signaling). For small cell numbers (7,000-15,000), the SuperSignal Western Blot Enhancer kit (Thermo Scientific) was used according to the manufacturer instructions. Signals were detected using the SuperSignal West Pico or SuperSignal West Femto chemiluminescence kits (Thermo Scientific). Blots were stripped with 0.2N NaOH and/or 1% SDS, 25mM glycine, pH=2 before re-probing.
Agathocleous M., Meacham C.E., Burgess R.J., Piskounova E., Zhao Z., Crane G.M., Cowin B.L., Bruner E., Murphy M.M., Chen W., Spangrude G.J., Hu Z., DeBerardinis R.J, & Morrison S.J. (2017). Ascorbate regulates haematopoietic stem cell function and leukaemogenesis. Nature, 549(7673), 476-481.
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10

Quantifying Histone Modifications by Western Blot

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Equal numbers of cells from each population were sorted into trichloroacetic acid (TCA, Sigma), and the TCA final concentration was adjusted to 10%. Samples were centrifuged at 16,000g for 15 minutes, and precipitates were washed twice with cold acetone and dried. Samples were solubilized in 9M urea, 2% Triton X-100, 1% DTT. LDS loading buffer (Life Technologies) was added. Samples were heated at 70°C for 10 minutes, separated on NuPAGE Bis-Tris polyacrylamide gels (Life Technologies), and transferred to 0.2μm PVDF membranes (BioRad) by wet transfer using NuPAGE transfer buffer (Life Technologies). Western blots were performed using antibodies against β-actin (AC-74, Sigma), histone H3 (polyclonal, Abcam, ab1791), H3K4me3 (C42D8, Cell Signaling), H3K27me3 (C36B11, Cell Signaling), H3K4me2 (C75H12, Cell Signaling) and H3K36me2 (C64G9, Cell Signaling). For small cell numbers (7,000-15,000), the SuperSignal Western Blot Enhancer kit (Thermo Scientific) was used according to the manufacturer instructions. Signals were detected using the SuperSignal West Pico or SuperSignal West Femto chemiluminescence kits (Thermo Scientific). Blots were stripped with 0.2N NaOH and/or 1% SDS, 25mM glycine, pH=2 before re-probing.
Agathocleous M., Meacham C.E., Burgess R.J., Piskounova E., Zhao Z., Crane G.M., Cowin B.L., Bruner E., Murphy M.M., Chen W., Spangrude G.J., Hu Z., DeBerardinis R.J, & Morrison S.J. (2017). Ascorbate regulates haematopoietic stem cell function and leukaemogenesis. Nature, 549(7673), 476-481.
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