Anti-phospho EZH2 (Thr345)

Histone-lysine N-methyltransferase EZH2 (UniProt: Q15910; also known as EC:2.1.1.356, ENX-1, Enhancer of zeste homolog 2, Lysine N-methyltransferase 6, EZH2) is encoded by the EZH2 (also known as KMT6) gene (Gene ID: 2146) in human. EZH2 is a polycomb group protein that is expressed in many tissues. It is the catalytic subunit of the PRC2/EED-EZH2 complex that methylates lysine 9 (H3K9me) and lysine 27 (H3K27me) of histone H3, leading to transcriptional repression of the affected target gene. It is shown to mono-, di- and trimethylate lysine 27 of histone H3 (H3K27me1, H3K27me2 and H3K27me3). It displays a preference for substrates with less methylation and loses activity when progressively more methyl groups are incorporated into H3K27, H3K27me0 > H3K27me1 > H3K27me2. EZH2 phosphorylation by Akt1 reduces its methyltransferase activity. Its phosphorylation at threonine 345 by CDK1 and CDK2 is shown to promote maintenance of H3K27me3 levels at EZH2-target loci, thus leading to epigenetic gene silencing. Compared to EZH1-containing complexes, it is more abundant in embryonic stem cells and plays a major role in forming H3K27me3, which is required for embryonic stem cell identity and proper differentiation. It is reported to regulate the circadian clock via histone methylation at the promoter of the circadian genes and is essential for the CRY1/2-mediated repression of the transcriptional activation of PER1/2 by the CLOCK-ARNTL/BMAL1 heterodimer. Overexpression of EZH2 has been reported in numerous tumors, including carcinomas of the breast, colon, larynx, testis, and lymphoma. Mutations in EZH2 gene are known to cause Weaver syndrome, a disease characterized by accelerated growth and osseous maturation and unusual craniofacial appearance. (Ref.: Chen, S., et al. (2010). Nat. Cell Biol. 12(11); 1108-1114; Etchegaray, J-P., et al. (2006). J. Biol. Chem. 281(30); 21209-21215; Cha, T-L., et al. (2005). Science. 310(5746); 306-310).

This rabbit polyclonal antibody specifically detects Histone-lysine N-methyltransferase EZH2 phosphorylated on threonine 345.

KLH-conjugated linear peptide corresponding to 12 amino acids surrounding phosphothreonine 345 from the internal region of human Histone-lysine N-methyltransferase EZH2.

Quality Control Testing

Evaluated by Western Blotting in LNCaP cell lysate.

Western Blotting Analysis (WB): A 1:1,000 dilution of this antibody detected EZH2 phosphorylated on threonine 345 in LNCaP cell lysate, but not in lysates treated with protein phosphatase (400 units for 30 min at 30°C).

Tested applications

Immunocytochemistry Analysis: A representative lot detected phospho EZH2 (Thr345) in Immunocytochemistry applications (Chen, S., et. al. (2010). Nat Cell Biol. 12(11):1108-14).

Note: Actual optimal working dilutions must be determined by end user as specimens, and experimental conditions may vary with the end user

Purified rabbit polyclonal antibody in buffer containing 0.1 M Tris-Glycine (pH 7.4), 150 mM NaCl with 0.05% sodium azide

0.1 mg/mL. Please refer to guidance on suggested starting dilutions and/or titers per application and sample type.

Recommended storage: +2°C to +8°C.

Concentration: Please refer to the Certificate of Analysis for the lot-specific concentration.

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