Revision 1
Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
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UniProt ID:

#Q15910

Entrez-Gene Id:

2146

Product Includes Quantity Reactivity MW(kDa) Isotype
Ezh2 (D2C9) XP® Rabbit mAb 5246 100 µl H M R Mk 98 Rabbit IgG
Phospho-Ezh2 (Thr311) (F1K1B) Rabbit mAb 70303 100 µl H M R Mk 98 Rabbit IgG

Please visit cellsignal.com for individual component applications, species cross-reactivity, dilutions, protocols, and additional product information.

Description

PhosphoPlus® Duets from Cell Signaling Technology (CST) provide a means to assess protein activation status. Each Duet contains an activation-state and total protein antibody to your target of interest. These CST® antibodies have been selected based upon superior performance in specified applications.

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/mL BSA, 50% glycerol, and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

Background

The polycomb group (PcG) proteins are involved in maintaining the silenced state of several developmentally regulated genes and contribute to the maintenance of cell identity, cell cycle regulation, and oncogenesis (1,2). Enhancer of zeste homolog 2 (Ezh2), a member of this large protein family, contains four conserved regions including domain I, domain II, and a cysteine-rich amino acid stretch that precedes the carboxy-terminal SET domain (3). The SET domain has been linked with histone methyltransferase (HMTase) activity. Moreover, mammalian Ezh2 is a member of a histone deacetylase complex that functions in gene silencing, acting at the level of chromatin structure (4). Ezh2 complexes methylate histone H3 at Lys9 and 27 in vitro, which is thought to be involved in targeting transcriptional regulators to specific loci (5). Ezh2 is deregulated in various tumor types, and its role, both as a primary effector and as a mediator of tumorigenesis, has become a subject of increased interest (6).
Ezh2 is phosphorylated on Thr311 by AMP-activated protein kinase (AMPK) in response to sustained energy starvation (7). Phosphorylation of Thr311 disrupts the interaction between Ezh2 and SUZ12, leading to attenuation of Ezh2 histone methyltransferase activity and suppression of oncogenic function (7). In addition, phosphorylation of Ezh2 on Thr311 correlates with better survival in ovarian and breast cancer patients (7).

  1. Sellers, W.R. and Loda, M. (2002) Cancer Cell 2, 349-50.
  2. Visser, H.P. et al. (2001) Br J Haematol 112, 950-8.
  3. Chen, H. et al. (1996) Genomics 38, 30-7.
  4. Tonini, T. et al. (2004) Oncogene 23, 4930-7.
  5. Müller, J. et al. (2002) Cell 111, 197-208.
  6. Kleer, C.G. et al. (2003) Proc Natl Acad Sci U S A 100, 11606-11.
  7. Wan, L. et al. (2018) Mol Cell 69, 279-291.e5.

Background References

    Trademarks and Patents

    Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
    PhosphoPlus is a registered trademark of Cell Signaling Technology, Inc.
    XP is a registered trademark of Cell Signaling Technology, Inc.
    All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

    限制使用

    除非 CST 的合法授书代表以书面形式书行明确同意,否书以下条款适用于 CST、其关书方或分书商提供的书品。 任何书充本条款或与本条款不同的客书条款和条件,除非书 CST 的合法授书代表以书面形式书独接受, 否书均被拒书,并且无效。

    专品专有“专供研究使用”的专专或专似的专专声明, 且未专得美国食品和专品管理局或其他外国或国内专管机专专专任何用途的批准、准专或专可。客专不得将任何专品用于任何专断或治专目的, 或以任何不符合专专声明的方式使用专品。CST 专售或专可的专品提供专作专最专用专的客专,且专用于研专用途。将专品用于专断、专防或治专目的, 或专专售(专独或作专专成)或其他商专目的而专专专品,均需要 CST 的专独专可。客专:(a) 不得专独或与其他材料专合向任何第三方出售、专可、 出借、捐专或以其他方式专专或提供任何专品,或使用专品制造任何商专专品,(b) 不得复制、修改、逆向工程、反专专、 反专专专品或以其他方式专专专专专品的基专专专或技专,或使用专品开专任何与 CST 的专品或服专专争的专品或服专, (c) 不得更改或专除专品上的任何商专、商品名称、徽专、专利或版专声明或专专,(d) 只能根据 CST 的专品专售条款和任何适用文档使用专品, (e) 专遵守客专与专品一起使用的任何第三方专品或服专的任何专可、服专条款或专似专专

    Revision 1
    #91910

    PhosphoPlus® Ezh2 (Thr311) Antibody Duet

    PhosphoPlus® Ezh2 (Thr311) Antibody Duet: Image 1 Expand Image
    使用 Ezh2 (D2C9) XP® Rabbit mAb #5246 对 MCF-7 细胞的裂解物 (0.1 mg/mL) 进行 Simple Western™ 分析。虚拟泳道式图像(左图)显示一抗稀释比例为 1:10 和 1:50 时的靶标条带(如图所示)。对应的电泳图(右图)为一抗稀释比例在 1:10(蓝线)和 1:50(绿线)时沿毛细血管内分子量的化学发光结果。在还原条件下,使用 66-440 kDa 分离模块在 ProteinSimple(BioTechne 品牌)的 Jess™ Simple Western 仪器上进行该实验。
    PhosphoPlus® Ezh2 (Thr311) Antibody Duet: Image 2 Expand Image
    使用 Ezh2 (D2C9) XP® Rabbit mAb 对 MCF7、Neuro-2a 和 COS-7 细胞系提取物进行蛋白质印迹分析。
    PhosphoPlus® Ezh2 (Thr311) Antibody Duet: Image 3 Expand Image
    在 Leica Bond Rx 上使用 Ezh2 (D2C9) XP® Rabbit mAb 对石蜡包埋的人结肠腺癌组织进行免疫组织化学分析。
    PhosphoPlus® Ezh2 (Thr311) Antibody Duet: Image 4 Expand Image
    CUT&Tag was performed with NCCIT cells and Ezh2 (D2C9) XP® Rabbit mAb, using CUT&Tag Assay Kit #77552. DNA library was prepared using CUT&Tag Dual Index Primers and PCR Master Mix for Illumina Systems #47415. The figure shows binding across HOXA1, a known target gene of Ezh2 (see our ChIP-qPCR figure).
    PhosphoPlus® Ezh2 (Thr311) Antibody Duet: Image 5 Expand Image
    Western blot analysis of extracts from OVCAR-8 cells, untreated (-) or treated with 2-Deoxy-D-glucose (10 mM, 6 hr; +), using Phospho-Ezh2 (Thr311) (F1K1B) Rabbit mAb (upper) or Ezh2 (D2C9) XP® Rabbit mAb #5246 (lower).
    PhosphoPlus® Ezh2 (Thr311) Antibody Duet: Image 6 Expand Image
    在 Leica BOND Rx 上使用 Ezh2 (D2C9) XP® Rabbit mAb 对石蜡包埋的人 B 细胞非霍奇金淋巴瘤进行免疫组织化学分析。
    PhosphoPlus® Ezh2 (Thr311) Antibody Duet: Image 7 Expand Image
    CUT&Tag was performed with NCCIT cells and Ezh2 (D2C9) XP® Rabbit mAb, using CUT&Tag Assay Kit #77552. DNA library was prepared using CUT&Tag Dual Index Primers and PCR Master Mix for Illumina Systems #47415. The figures show binding across the HOXA gene cluster (upper), and the HOXD gene cluster (lower), known target genes of Ezh2 (see our ChIP-qPCR figure).
    PhosphoPlus® Ezh2 (Thr311) Antibody Duet: Image 8 Expand Image
    Western blot analysis of extracts from C2C12 cells, untreated (-) or treated with A-769662 #64839 (1 μM, 1 hr; +), using Phospho-Ezh2 (Thr311) (F1K1B) Rabbit mAb (upper) or Ezh2 (D2C9) XP® Rabbit mAb #5246 (lower).
    PhosphoPlus® Ezh2 (Thr311) Antibody Duet: Image 9 Expand Image
    使用 Ezh2 (D2C9) XP® Rabbit mAb 对石蜡包埋的人乳腺癌进行免疫组织化学分析。
    PhosphoPlus® Ezh2 (Thr311) Antibody Duet: Image 10 Expand Image
    Western blot analysis of extracts from H-4-II-E and COS-7 cells using Phospho-Ezh2 (Thr311) (F1K1B) Rabbit mAb.
    PhosphoPlus® Ezh2 (Thr311) Antibody Duet: Image 11 Expand Image
    使用 Ezh2 (D2C9) XP® Rabbit mAb 对石蜡包埋的人宫颈癌进行免疫组织化学分析。
    PhosphoPlus® Ezh2 (Thr311) Antibody Duet: Image 12 Expand Image
    使用 Ezh2 (D2C9) XP® Rabbit mAb 对石蜡包埋的人淋巴瘤进行免疫组织化学分析。
    PhosphoPlus® Ezh2 (Thr311) Antibody Duet: Image 13 Expand Image
    使用 Ezh2 (D2C9) XP® 兔单克隆抗体对石蜡包埋的 4T1 小鼠同基因肿瘤进行免疫组织化学分析。
    PhosphoPlus® Ezh2 (Thr311) Antibody Duet: Image 14 Expand Image
    使用 Ezh2 (D2C9) XP® Rabbit mAb(绿色)对小鼠海马(左图)和小脑(右图)进行共聚焦免疫荧光分析。肌动蛋白微丝由 DyLight 554 Phalloidin #13054(红色)标记。蓝色伪彩 = DRAQ5® #4084(DNA 荧光染料)。
    PhosphoPlus® Ezh2 (Thr311) Antibody Duet: Image 15 Expand Image
    使用 Ezh2 (D2C9) XP® Rabbit mAb(绿色)和S6 Ribosomal Protein (54D2) Mouse mAb #2317(蓝色)对 HeLa 细胞进行共聚焦免疫荧光分析。使用 DY-554 phalloidin(红色)标记肌动蛋白纤丝。
    PhosphoPlus® Ezh2 (Thr311) Antibody Duet: Image 16 Expand Image
    使用 Ezh2 (D2C9) XP® Rabbit mAb 对石蜡包埋的 小鼠大脑进行免疫组织化学分析。
    PhosphoPlus® Ezh2 (Thr311) Antibody Duet: Image 17 Expand Image
    使用 EZH2 (D2C9) XP® Rabbit mAb 对未经处理(左图)和经抗人 CD3(10ug/ml,涂层平板)和抗人 CD28 (5ug/ml) 在 37ºC 下处理(右图)337 天的人外周血单核细胞进行流式细胞分析,并与抗人 CD3 抗体联合染色。使用 Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor 488 Conjugate) #4412 作为二抗。
    PhosphoPlus® Ezh2 (Thr311) Antibody Duet: Image 18 Expand Image
    使用 Ezh2 (D2C9) XP® Rabbit mAb 对石蜡包埋的小鼠肝脏进行免疫组织化学分析。
    PhosphoPlus® Ezh2 (Thr311) Antibody Duet: Image 19 Expand Image
    使用 SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003,对 HeLa 细胞中提取的交联染色质,在加入 Ezh2 (D2C9) XP® Rabbit mAb 或 Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAb 后,进行染色质免疫沉淀。使用 DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795 制备 DNA 库。已发现 EZH2 和 H3K27me3 在染色质中相互结合。该图显示了 EZH2 和 H3K27me3 在 MYT1 基因中的结合。欲知其他 ChIP-seq 情况,请下载产品说明书。
    PhosphoPlus® Ezh2 (Thr311) Antibody Duet: Image 20 Expand Image
    使用 Ezh2 (D2C9) XP® Rabbit mAb 对石蜡包埋的小鼠睾丸进行免疫组织化学分析。
    PhosphoPlus® Ezh2 (Thr311) Antibody Duet: Image 21 Expand Image
    使用 SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003,对 HeLa 细胞中提取的交联染色质,在加入 Ezh2 (D2C9) XP® Rabbit mAb 或 Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAb 后,进行染色质免疫沉淀。使用 DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795 制备 DNA 库。已发现 EZH2 和 H3K27me3 在染色质中相互结合。结果图显示在染色体 20(上图)内 EZH2 和 H3K27me3 两者的结合,包括 MYT1 基因(下图)。
    PhosphoPlus® Ezh2 (Thr311) Antibody Duet: Image 22 Expand Image
    使用 SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003 对 NCCIT 细胞中提取的交联染色质,在加入 Ezh2 (D2C9) XP® Rabbit mAb 或 Normal Rabbit IgG #2729 后,进行染色质免疫沉淀。使用 SimpleChIP® Human HoxA1 Intron 1 Primers #7707、SimpleChIP® Human HoxA2 Promoter Primers #5517 和 SimpleChIP® Human α Satellite Repeat Primers #4486 通过实时 PCR 对富集 DNA 进行定量分析。将每份样品中免疫沉淀的 DNA 的量表现为相对于所输入染色质总量(等于 1)的信号。
    PhosphoPlus® Ezh2 (Thr311) Antibody Duet: Image 23 Expand Image
    使用 CUT&RUN Assay Kit #86652,通过 NCCIT 细胞和 Ezh2 (D2C9) XP® Rabbit mAb 进行 CUT&RUN。使用 DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795 制备 DNA 库。结果图显示在 Ezh2 的已知靶标基因 HoxA1 处结合(参见包含 CUT&RUN-qPCR 数据的其他结果图)。
    PhosphoPlus® Ezh2 (Thr311) Antibody Duet: Image 24 Expand Image
    使用 CUT&RUN Assay Kit #86652,通过 NCCIT 细胞和 Ezh2 (D2C9) XP® Rabbit mAb 进行 CUT&RUN。使用 DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795 制备 DNA 库。结果图显示在染色体 7(上图)内的结合,包括 HoxA 基因(下图),HoxA 基因是 EZH2 靶标基因的已知基因簇(参见包含 CUT&RUN-qPCR 数据的其他结果图)。
    PhosphoPlus® Ezh2 (Thr311) Antibody Duet: Image 25 Expand Image
    使用 CUT&RUN Assay Kit #86652,NCCIT 细胞和 Ezh2 (D2C9) XP® Rabbit mAb 或 Rabbit (DA1E) mAb IgG XP® Isotype Control (CUT&RUN) #66362 进行 CUT&RUN。使用 SimpleChIP® Human HoxA1 Intron 1 Primers #7707、SimpleChIP® Human HoxA2 Promoter Primers #5517 和 SimpleChIP® Human α Satellite Repeat Primers #4486 通过实时 PCR 对富集 DNA 进行定量分析。将每份样品中免疫沉淀的 DNA 的量表现为相对于所输入染色质总量(等于 1)的信号。