Anti ezh2 d2c9

Manufactured by Cell Signaling Technology

Anti-EZH2 (D2C9) is a rabbit monoclonal antibody that recognizes the EZH2 protein. EZH2 is a histone methyltransferase that plays a role in the epigenetic regulation of gene expression.

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6 protocols using anti ezh2 d2c9

Comprehensive Protein Expression Analysis

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Cells were lysed in RIPA lysis buffer (50 mM Tris-HCL [pH 8], 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, protease inhibitors) for 30 min at 4°C. Protein extracts were resolved by SDS-PAGE and blotted to Nitrocellulose membranes and probed with the following antibodies: anti-beta Actin (Sigma-Aldrich, A5441), anti–EZH2 (AC22, Cell Signaling Technology, 3147S), anti-EZH2 (D2C9, Cell Signaling, 5246), anti-pT487 EZH2 (Abcam, ab109398), anti–SUZ12 (Cell Signaling Technology, 3737S), anti–EED (Millipore, 09-774), anti-EZH1 (Abcam, ab13665), anti-total H3 (Abcam, ab39763), anti-H3K27me3 (Cell Signaling Technologies, 9733), anti-V5 (Abcam, ab9116), anti-total CDK1 (Cell Signaling Technologies, 9112S), anti-pCDK1 (Cell Signaling Technologies, 9111S), anti-MRP1/ABCC1 (Santa Cruz, sc-18835), anti-Ubiquitin (Abcam, ab7780), anti-HOXB7 (Abcam, ab51237), anti-HOXA9 (Abcam, ab140631), anti-TRIM21 antibody (Abcam, ab91432), anti-HSP90 antibody (Abcam, ab13495) and anti-STIP1 antibody (Abcam, ab126724).
Densitometric analysis was performed using ImageJ software (NIH, Bethesda, MD).

Göllner S., Oellerich T., Agrawal-Singh S., Schenk T., Klein H.U., Rohde C., Pabst C., Sauer T., Lerdrup M., Tavor S., Stölzel F., Herold S., Ehninger G., Köhler G., Pan K.T., Urlaub H., Serve H., Dugas M., Spiekermann K., Vick B., Jeremias I., Berdel W.E., Hansen K., Zelent A., Wickenhauser C., Müller L.P., Thiede C, & Müller-Tidow C. (2016). Loss of the Histone Methyltransferase EZH2 induces Resistance to Multiple Drugs in Acute Myeloid Leukemia. Nature medicine, 23(1), 69-78.

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Immunofluorescence Microscopy of Nuclear Proteins

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For immunofluorescence microscopy, cells were cytospinned on glass slides and subsequently fixed using 4% paraformaldehyde in PBS. Subsequently, cells were permeabilized using PBS containing 0.1% Triton X-100. Primary antibodies include anti-Fibrillarin (ab5821, Abcam), anti-NPM1 (FC-61991, Thermo Fisher Scientific), anti-CBX4 (E6L7X, Cell Signalling Technology) and anti-EZH2 (D2C9, Cell Signalling Technology) followed by secondary antibody staining using Alexa Fluor 488 goat-anti-rabbit (Thermo Fisher Scientific, A-11008), Alexa Fluor 488 goat-anti-mouse (Thermo Fisher Scientific, A-11001), Alexa Fluor 647 goat-anti-rabbit (Thermo Fisher Scientific, A-21244), or Alexa Fluor 647 goat-anti-mouse (Thermo Fisher Scientific, A-21235). Images were acquired on a Leica DM6000B microscope using a 40x dry objective (HCX PL FLUOTAR, numerical aperture: 0.75) or a 63x immersion objective (PL S-APO, numerical aperture: 1.30) using LAS-AF software (Leica). Confocal images were acquired on a Leica TCS SP8 confocal laser-scanning microscope using a HC PL APO CS2 63x/1.4 oil objective, and excitation with 488 nm (20 mW) and 633 nm (30 mW) laser lines.

Azkanaz M., Rodríguez López A., de Boer B., Huiting W., Angrand P.O., Vellenga E., Kampinga H.H., Bergink S., Martens J.H., Schuringa J.J, & van den Boom V. (2019). Protein quality control in the nucleolus safeguards recovery of epigenetic regulators after heat shock. eLife, 8, e45205.

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ChIP Analysis Protocol for Epigenetic Markers

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ChIP analysis was essentially performed as described previously (Frank et al., 2001 (link)). ChIP reactions were performed using the following antibodies: anti-GFP (ab290, Abcam), anti-CBX8 (C15410333, Diagenode), anti-EZH2 (D2C9, Cell Signaling), anti-H2AK119ub (D27C4, Cell Signaling), anti-H3K27me3 (C15410195, Diagenode), anti-H3K27Ac (C15410196, Diagenode), anti-H3K4me1 (C15410194, Diagenode), and anti-H3K4me3 (C15410003, Diagenode). ChIP efficiencies were assessed using qPCR. Primer sequences can be found in Supplementary file 6.

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Comprehensive Protein Expression Analysis

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Western blot analysis was performed as published previously (van den Boom et al., 2013 (link)). The following antibodies were used: anti-GFP (ab290, Abcam), anti-EZH2 (D2C9, Cell Signaling Technology), anti-SUZ12, anti-CBX4 (09–029, Merck), anti-CBX8 (C15410333, Diagenode), anti-RING1B (ab181140, Abcam), anti-BMI1 (F6, Merck), anti-DNAJB1 (SPA-400, Enzo Life Sciences), anti-HSP70 (SPA-810, Enzo Life Sciences), anti-Fibrillarin (ab5821, Abcam), anti-H3K27me3 (07–449, Merck), anti-H2AK119ub (D27C4, Cell Signaling Technology), anti-H3K4me3 (ab8580, Abcam), and anti-β-Actin (C4, Santa Cruz).

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Prostate Cancer Cell Line Characterization

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The AR expressing, via lentiviral infection of LV-AR, hTERT immortalized human prostate epithelial 957E/hTERT cells (PrEC-AR) and c-Myc transformed PrEC-AR cells (PrEC-AR-c-Myc) were from Dr. John T. Issacs (Johns Hopkins University). Hi-MYC cell lines were purchased from AACR and cultured in DMEM supplemented with 10% FBS. PrEC-AR and PrEC-AR-c-Myc cell lines were cultured in Keratinocyte Serum Free Media supplemented with supplemented with bovine pituitary extract and recombinant epidermal growth factor (ThermoFisher) and 1% penicillin/streptomycin in 5% CO2 environment at 37°C. For in vitro experiments DZNep, GSK126, and EPZ6438 were all purchased from Xcessbio. For in vivo experiments DZNep was purchased from Sigma, and Epizyme Pharmaceuticals generously supplied EPZ011989. Antibodies used were anti-P53 (D2H9O, Cell Signaling), anti-EZH2 (D2C9, Cell Signaling), anti-H3K27me3 (C36B11, Cell Signaling), and anti-Ki67 (Sp6, Thermo Fisher). Mycoplasma testing is conducted every 3 months using standard PCR protocol from Sigma: Venor GeM Mycoplasma Detection Kit. Cell lines are authenticated using standard STR profiling.

Burkhart D.L., Morel K.L., Wadosky K.M., Labbé D.P., Galbo P.M., Dalimov Z., Xu B., Loda M, & Ellis L. (2020). Evidence that EZH2 Deregulation is an Actionable Therapeutic Target for Prevention of Prostate Cancer. Cancer prevention research (Philadelphia, Pa.), 13(12), 979-988.

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EZH2, DNMT Interactions and Stability

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The full-length cDNA of Ezh2 was subcloned into pcDNA3.1-HA vector and Dnmt1, Dnmt3a, Dnmt3b were subcloned into pcDNA3.1-FLAG vector for expression. For protein stability studies, the pcDNA3.1-EZH2-HA vector was co-transfected with pcDNA3.1-FLAG-DNMT1 and pcDNA3.1-FLAG-DNMT3B vectors into 293T cells by using TranIT-293 Transfection Reagent (MIR 2705, Mirus), respectively. After 24 h, the transfected cells were treated with cycloheximide for 24 h, and the working solution of cycloheximide was 200 μg/mL. For immunoprecipitation studies, the 293T cells were transfected with pcDNA3.1-FLAG-DNMT1, pcDNA3.1-FLAG-DNMT3A, pcDNA3.1-FLAG-DNMT3B, pcDNA3.1-FLAG vectors. Cells were harvested 48 h after transfection, the extracted product were incubated with 2 μg of anti-Ezh2 (D2C9, Cell Signaling Technology), 2 μg of anti-FLAG (M2; Sigma-Aldrich) for 5 h, and then incubated with Dynabeads Protein G (10004D; Life Technologies) for 2 h. After washing, the protein stability and immunoprecipitated samples were analyzed by immunoblot analysis with appropriate antibodies.

Luo Y., Li D., Xie L., Lei S., Chen X., Wang C., Yao D., Li L., Fang J., Chen C., Yuan S., Li F., Xie X., Zhang Y., Li Z., Hu L., Tang J., Ye L., Wei Z, & He R. (2022). EZH2 restricts Tcf7 DNA methylation and promotes TFH differentiation during acute viral infection. Frontiers in Immunology, 13, 942465.

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