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80780
PhosphoPlus® Ezh2 (Thr345) Antibody Duet
Primary Antibodies
Antibody Duet
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PhosphoPlus® Ezh2 (Thr345) Antibody Duet #80780

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Simple Western™ analysis of lysates (0.1 mg/mL) from MCF-7 cells using Ezh2 (D2C9) XP® Rabbit mAb #5246. The virtual lane view (left) shows the target band (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ ​​​​​​​ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 66 – 440 kDa separation module.
Immunoprecipitation of phospho-Ezh2 (Thr345) protein from NCCIT cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Phospho-Ezh2 (Thr345) (E7T7K) Rabbit mAb. Western blot analysis was performed using Phospho-Ezh2 (Thr345) (E7T7K) Rabbit mAb. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as a secondary antibody.
Western blot analysis of extracts from HCT 116 cells, untreated (-) or treated with λ phosphatase and calf intestinal phosphatase (CIP) (+), using Phospho-Ezh2 (Thr345) (E7T7K) Rabbit mAb (upper) or Ezh2 (D2C9) XP® Rabbit mAb #5246 (lower).
CUT&RUN was performed with NCCIT cells and Phospho-Ezh2 (Thr345) (E7T7K) Rabbit mAb using CUT&RUN Assay Kit #86652. DNA library was prepared using DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795. The figure shows binding across NR2E1, a known target gene of Ezh2 (see additional figure containing CUT&RUN-qPCR data).
Chromatin immunoprecipitations were performed with cross-linked chromatin from NCCIT cells and either Phospho-Ezh2 (Thr345) (E7T7K) Rabbit mAb or Ezh2 (D2C9) XP® Rabbit mAb #5246, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795. The figure shows binding across HoxA, a known target gene of Ezh2 (see additional figure containing ChIP-qPCR data).
Western blot analysis of extracts from MCF7, Neuro-2a, and COS-7 cell lines using Ezh2 (D2C9) XP® Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human colon adenocarcinoma using Ezh2 (D2C9) XP® Rabbit mAb performed on the Leica BOND Rx.
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).
Western blot analysis of extracts from Neuro-2a, H-4-II-E, and COS-7 cells using Phospho-Ezh2 (Thr345) (E7T7K) Rabbit mAb.
CUT&RUN was performed with NCCIT cells and Phospho-Ezh2 (Thr345) (E7T7K) Rabbit mAb using CUT&RUN Assay Kit #86652. DNA library was prepared using DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795. The figures show binding across chromosome 6 (upper), including NR2E1 (lower), a known target gene of Ezh2 (see additional figure containing CUT&RUN-qPCR data).
Chromatin immunoprecipitations were performed with cross-linked chromatin from NCCIT cells and either Phospho-Ezh2 (Thr345) (E7T7K) Rabbit mAb or Ezh2 (D2C9) XP® Rabbit mAb #5246, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795. The figures show binding across HoxA (upper) and HoxD (lower), known target genes of Ezh2 (see additional figure containing ChIP-qPCR data).
Immunohistochemical analysis of paraffin-embedded human B-cell non-Hodgkin lymphoma using Ezh2 (D2C9) XP® Rabbit mAb performed on the Leica BOND Rx.
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).
CUT&RUN was performed with NCCIT cells and either Phospho-Ezh2 (Thr345) (E7T7K) Rabbit mAb or Rabbit (DA1E) mAb IgG XP® Isotype Control (CUT&RUN) #66362, using CUT&RUN Assay Kit #86652. The enriched DNA was quantified by real-time PCR using human NR2E1 promoter primers and human BRD2 upstream primers. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.
Chromatin immunoprecipitations were performed with cross-linked chromatin from NCCIT cells and either Phospho-Ezh2 (Thr345) (E7T7K) Rabbit mAb or Normal Rabbit IgG #2729 using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human HoxA1 Intron 1 Primers #7707, SimpleChIP® Human HoxA2 Promoter Primers #5517, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.
Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Ezh2 (D2C9) XP® Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human cervical carcinoma using Ezh2 (D2C9) XP® Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human lymphoma using Ezh2 (D2C9) XP® Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded 4T1 mouse syngeneic tumor using Ezh2 (D2C9) XP® Rabbit mAb.
Confocal immunofluorescent analysis of mouse hippocampus (left) and cerebellum (right) using Ezh2 (D2C9) XP® Rabbit mAb (green). Actin filaments were labeled with DyLight™ 554 Phalloidin #13054 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Confocal immunofluorescent analysis of HeLa cells using Ezh2 (D2C9) XP® Rabbit mAb (green) and S6 Ribosomal Protein (54D2) Mouse mAb #2317 (blue). Actin filaments were labeled with DY-554 phalloidin (red).
Immunohistochemical analysis of paraffin-embedded mouse brain using Ezh2 (D2C9) XP® Rabbit mAb.
Flow cytometric analysis of human peripheral blood mononuclear cells untreated (left) and treated (right) with anti-human CD3 (10ug/ml, coated plates) and anti-human CD28 (5ug/ml) for 3 days at 37ºC using EZH2 (D2C9) XP® Rabbit mAb and co-stained with an anti-human CD3 antibody. Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor 488 Conjugate) #4412 was used as a secondary antibody.
Immunohistochemical analysis of paraffin-embedded mouse liver using Ezh2 (D2C9) XP® Rabbit mAb.
Chromatin immunoprecipitations were performed with cross-linked chromatin from Hela cells and either Ezh2 (D2C9) XP® Rabbit mAb or Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAb, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. EZH2 and H3K27me3 are known to associate with each other on chromatin. The figure shows binding of both EZH2 and H3K27me3 across the MYT1 gene. For additional ChIP-seq tracks, please download the product datasheet.
Immunohistochemical analysis of paraffin-embedded mouse testis using Ezh2 (D2C9) XP® Rabbit mAb.
Chromatin immunoprecipitations were performed with cross-linked chromatin from Hela cells and either Ezh2 (D2C9) XP® Rabbit mAb or Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAb, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. EZH2 and H3K27me3 are known to associate with each other on chromatin. The figure shows binding of both EZH2 and H3K27me3 across chromosome 20 (upper), including MYT1 gene (lower).
Chromatin immunoprecipitations were performed with cross-linked chromatin from NCCIT cells and either Ezh2 (D2C9) XP® Rabbit mAb or Normal Rabbit IgG #2729 using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human HoxA1 Intron 1 Primers #7707, SimpleChIP® Human HoxA2 Promoter Primers #5517, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.
CUT&RUN was performed with NCCIT cells and Ezh2 (D2C9) XP® Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figures show binding across HoxA1, a known target gene of Ezh2 (see additional figure containing CUT&RUN-qPCR data).
CUT&RUN was performed with NCCIT cells and Ezh2 (D2C9) XP® Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figures show binding across chromosome 7 (upper), including HoxA genes (lower), a known cluster of EZH2 target genes (see additional figure containing CUT&RUN-qPCR data).
CUT&RUN was performed with NCCIT cells and either Ezh2 (D2C9) XP® Rabbit mAb or Rabbit (DA1E) mAb IgG XP® Isotype Control (CUT&RUN) #66362, using CUT&RUN Assay Kit #86652. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human HoxA1 Intron 1 Primers #7707, SimpleChIP® Human HoxA2 Promoter Primers #5517, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.
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To Purchase # 80780
Cat. # Size Qty. Price Inventory
80780S		 1 Kit

Product Includes Quantity Reactivity MW(kDa) Isotype
Ezh2 (D2C9) XP® Rabbit mAb 5246 100 µl H M R Mk 98 Rabbit IgG
Phospho-Ezh2 (Thr345) (E7T7K) Rabbit mAb 45153 100 µl H M R Mk 98 Rabbit IgG

Product 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.

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 was shown to be phosphorylated by cyclin-dependent kinase 1 (CDK1) at Thr345 and Thr487 in a cell cycle-dependent manner (7). It has also been shown that the ANCR long non-coding RNA potentiates the Ezh2-CDK1 interaction, leading to increased phosphorylation of Thr345 and Thr487 on Ezh2 and facilitating ubiquitination and subsequent degradation of this protein (8).

Pathways

Explore pathways related to this product.

Limited Uses

Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

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