Phospho-EZH2 (Thr416) Antibody - #AF3585

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Product: Phospho-EZH2 (Thr416) Antibody
Catalog: AF3585
Description: Rabbit polyclonal antibody to Phospho-EZH2 (Thr416)
Application: IHC IF/ICC
Reactivity: Human, Mouse, Rat
Mol.Wt.: 85kD, 100kD; 85kD(Calculated).
Uniprot: Q15910
RRID: AB_2846899

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MSDS
Data Sheet
COA
Protocols
WB Handbook
IHC Protocol
IF/ICC Protocol

Product Info

Source:
Rabbit
Application:
IHC 1:50-1:200, IF/ICC 1:100-1:500
*The optimal dilutions should be determined by the end user.
*Tips:

WB: For western blot detection of denatured protein samples. IHC: For immunohistochemical detection of paraffin sections (IHC-p) or frozen sections (IHC-f) of tissue samples. IF/ICC: For immunofluorescence detection of cell samples. ELISA(peptide): For ELISA detection of antigenic peptide.

Reactivity:
Human,Mouse,Rat
Clonality:
Polyclonal
Specificity:
Phospho-EZH2 (Thr416) Antibody detects endogenous levels of EZH2 only when phosphorylated at Thr416.
RRID:
AB_2846899
Cite Format: Affinity Biosciences Cat# AF3585, RRID:AB_2846899.
Conjugate:
Unconjugated.
Purification:
The antibody is from purified rabbit serum by affinity purification via sequential chromatography on phospho-peptide and non-phospho-peptide affinity columns.
Storage:
Rabbit IgG in phosphate buffered saline , pH 7.4, 150mM NaCl, 0.02% sodium azide and 50% glycerol. Store at -20 °C. Stable for 12 months from date of receipt.
Alias:

Fold/Unfold

Enhancer of zeste 2; enhancer of zeste 2 polycomb repressive complex 2 subunit; Enhancer of zeste homolog 2 (Drosophila); Enhancer of zeste homolog 2; Enhancer of zeste, Drosophila, homolog 2; ENX 1; Enx 1h; ENX-1; ENX1; Enx1h; EZH 2; EZH1; EZH2; EZH2_HUMAN; EZH2b; Histone-lysine N-methyltransferase EZH2; KMT 6; KMT6; KMT6A; Lysine N-methyltransferase 6; MGC9169; WVS; WVS2;

Immunogens

Immunogen:

A synthesized peptide derived from human EZH2 around the phosphorylation site of Thr416.

Uniprot:

Q15910(EZH2_HUMAN) >>Visit HPA database.

Gene(ID):
EZH2(2146)
Expression:
Q15910 EZH2_HUMAN:

Expressed in many tissues. Overexpressed in numerous tumor types including carcinomas of the breast, colon, larynx, lymphoma and testis.

Sequence:
MGQTGKKSEKGPVCWRKRVKSEYMRLRQLKRFRRADEVKSMFSSNRQKILERTEILNQEWKQRRIQPVHILTSVSSLRGTRECSVTSDLDFPTQVIPLKTLNAVASVPIMYSWSPLQQNFMVEDETVLHNIPYMGDEVLDQDGTFIEELIKNYDGKVHGDRECGFINDEIFVELVNALGQYNDDDDDDDGDDPEEREEKQKDLEDHRDDKESRPPRKFPSDKIFEAISSMFPDKGTAEELKEKYKELTEQQLPGALPPECTPNIDGPNAKSVQREQSLHSFHTLFCRRCFKYDCFLHPFHATPNTYKRKNTETALDNKPCGPQCYQHLEGAKEFAAALTAERIKTPPKRPGGRRRGRLPNNSSRPSTPTINVLESKDTDSDREAGTETGGENNDKEEEEKKDETSSSSEANSRCQTPIKMKPNIEPPENVEWSGAEASMFRVLIGTYYDNFCAIARLIGTKTCRQVYEFRVKESSIIAPAPAEDVDTPPRKKKRKHRLWAAHCRKIQLKKDGSSNHVYNYQPCDHPRQPCDSSCPCVIAQNFCEKFCQCSSECQNRFPGCRCKAQCNTKQCPCYLAVRECDPDLCLTCGAADHWDSKNVSCKNCSIQRGSKKHLLLAPSDVAGWGIFIKDPVQKNEFISEYCGEIISQDEADRRGKVYDKYMCSFLFNLNNDFVVDATRKGNKIRFANHSVNPNCYAKVMMVNGDHRIGIFAKRAIQTGEELFFDYRYSQADALKYVGIEREMEIP

PTMs - Q15910 As Substrate

Site PTM Type Enzyme
K7 Ubiquitination
S8 Phosphorylation
K10 Ubiquitination
S21 Phosphorylation P31749 (AKT1)
Y23 Phosphorylation
K39 Ubiquitination
K61 Ubiquitination
S75 O-Glycosylation
S76 O-Glycosylation
S76 Phosphorylation
S212 Phosphorylation
S220 Phosphorylation
K234 Acetylation
K234 Ubiquitination
Y244 Phosphorylation
T261 Phosphorylation
S277 Phosphorylation
T302 Phosphorylation
T305 Phosphorylation
T311 Phosphorylation
K318 Ubiquitination
T339 Phosphorylation
T345 Phosphorylation P24941 (CDK2) , P06493 (CDK1)
K348 Acetylation
S362 Phosphorylation
S363 Phosphorylation P49841 (GSK3B)
S366 Phosphorylation
T367 Phosphorylation P49841 (GSK3B) , Q16539 (MAPK14)
T369 Phosphorylation
S375 Phosphorylation
S380 Phosphorylation
T388 Phosphorylation
S405 Phosphorylation
S406 Phosphorylation
S408 Phosphorylation
S412 Phosphorylation
T416 Phosphorylation P24941 (CDK2)
T460 Phosphorylation
K461 Ubiquitination
K472 Ubiquitination
S474 Phosphorylation
S475 Phosphorylation
T487 Phosphorylation P06493 (CDK1)
K505 Methylation
K509 Methylation
K510 Methylation
K569 Ubiquitination
K597 Ubiquitination
K602 Ubiquitination
K629 Ubiquitination
K634 Ubiquitination
Y641 Phosphorylation O60674 (JAK2)
S690 Phosphorylation
Y696 Phosphorylation
K713 Ubiquitination
T718 Phosphorylation
S729 Phosphorylation
K735 Methylation
K735 Ubiquitination

Research Backgrounds

Function:

Polycomb group (PcG) protein. Catalytic subunit of the PRC2/EED-EZH2 complex, which methylates 'Lys-9' (H3K9me) and 'Lys-27' (H3K27me) of histone H3, leading to transcriptional repression of the affected target gene. Able to mono-, di- and trimethylate 'Lys-27' of histone H3 to form H3K27me1, H3K27me2 and H3K27me3, respectively. Displays a preference for substrates with less methylation, loses activity when progressively more methyl groups are incorporated into H3K27, H3K27me0 > H3K27me1 > H3K27me2. 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. The PRC2/EED-EZH2 complex may also serve as a recruiting platform for DNA methyltransferases, thereby linking two epigenetic repression systems. Genes repressed by the PRC2/EED-EZH2 complex include HOXC8, HOXA9, MYT1, CDKN2A and retinoic acid target genes. EZH2 can also methylate non-histone proteins such as the transcription factor GATA4 and the nuclear receptor RORA. Regulates the circadian clock via histone methylation at the promoter of the circadian genes. Essential for the CRY1/2-mediated repression of the transcriptional activation of PER1/2 by the CLOCK-ARNTL/BMAL1 heterodimer; involved in the di and trimethylation of 'Lys-27' of histone H3 on PER1/2 promoters which is necessary for the CRY1/2 proteins to inhibit transcription.

PTMs:

Phosphorylated by AKT1. Phosphorylation by AKT1 reduces methyltransferase activity. Phosphorylation at Thr-345 by CDK1 and CDK2 promotes maintenance of H3K27me3 levels at EZH2-target loci, thus leading to epigenetic gene silencing.

Sumoylated.

Glycosylated: O-GlcNAcylation at Ser-75 by OGT increases stability of EZH2 and facilitates the formation of H3K27me3 by the PRC2/EED-EZH2 complex.

Subcellular Location:

Nucleus.

Extracellular region or secreted Cytosol Plasma membrane Cytoskeleton Lysosome Endosome Peroxisome ER Golgi apparatus Nucleus Mitochondrion Manual annotation Automatic computational assertionSubcellular location
Tissue Specificity:

Expressed in many tissues. Overexpressed in numerous tumor types including carcinomas of the breast, colon, larynx, lymphoma and testis.

Subunit Structure:

Binds ATRX via the SET domain (Probable). Component of the PRC2/EED-EZH2 complex, which includes EED, EZH2, SUZ12, RBBP4 and RBBP7 and possibly AEBP2. The minimum components required for methyltransferase activity of the PRC2/EED-EZH2 complex are EED, EZH2 and SUZ12. The PRC2 complex may also interact with DNMT1, DNMT3A, DNMT3B and PHF1 via the EZH2 subunit and with SIRT1 via the SUZ12 subunit. Interacts with HDAC1 and HDAC2. Interacts with PRAME. Interacts with CDYL. Interacts with CLOCK, ARNTL/BMAL1 and CRY1 (By similarity). Interacts with DNMT3L; the interaction is direct (By similarity). Interacts with EZHIP; the interaction blocks EZH2 methyltransferase activity.

Family&Domains:

Belongs to the class V-like SAM-binding methyltransferase superfamily. Histone-lysine methyltransferase family. EZ subfamily.

Research Fields

· Human Diseases > Cancers: Overview > MicroRNAs in cancer.

· Metabolism > Amino acid metabolism > Lysine degradation.

References

1). Activation of Bivalent Gene POU4F1 Promotes and Maintains Basal-like Breast Cancer. Advanced science (Weinheim, Baden-Wurttemberg, Germany), 2024 (PubMed: 38491910) [IF=15.1]

Application: WB    Species: Human    Sample: MDA‐MB‐231 and BT549 cells

Figure 5 POU4F1 repressed ERα expression through CDK2/EZH2 axis. A) ChIP‐qPCR analysis of the enrichment of POU4F1 at the promoter of ESR1 in MDA‐MB‐231 and BT549 cells. B,C) Indicated protein and mRNA expression in MDA‐MB‐231 and BT549 transfected with POU4F1 siRNAs and control siRNA. D, E) ChIP‐qPCR analysis of the enrichment of EZH2 and H3K27me3 at the promoter of ESR1 in MDA‐MB‐231 treated with DMSO, 100 nM SNS‐032 and 25 nM Dinaciclib D) and 5 µM GSK126 and 10 µM EPZ‐6438 E) for 72 h. F,G) The mRNA expression of ESR1 and its downstream genes in MDA‐MB‐231 cells treated with DMSO, 100 nM SNS‐032 and 25 nM Dinaciclib F) and 5 µM GSK126 and 10 µM EPZ‐6438 G) for 72 h. H) ChIP‐qPCR analysis of the enrichment of EZH2 and H3K27me3 at the promoter of ESR1 in POU4F1‐KO MDA‐MB‐231 cells. I) Western blotting showing the expression of indicated protein after rescue with CDK2, EZH2 and POU4F1 plasmid in POU4F1‐KO MDA‐MB‐231 cells. Data were presented as mean ± S.D., n = 3 A, B, D‐H). ns, no significance, *P < 0.05, **P < 0.01, ***P < 0.001 compared with IgG A),siCtrl B) and DMSO F,G) by two‐tailed Student's t‐test A) and two‐tailed one‐way ANOVA and Dunnett's B,F,G) or Bonferroni multiple‐comparisons test.

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