Phospho-Histone H2A.X (Ser139)

2577:

Flow , IHC / Paraffin , Immunofluorescence , Western Blotting

Phospho-H2A.X (Ser139) Antibody detects endogenous levels of H2A.X only when phosphorylated at Ser139.

Antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser139 of human H2A.X.

Western Blotting

Western blot analysis of extracts from 293 cells, untreated or UV-treated, using Phospho-Histone H2A.X (Ser139) Antibody (upper) or Histone H2A Antibody #2572 (lower).

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded HT-29 cells untreated (left) or treated (right) with UV (upper) or doxorubicin (lower) using Phospho-Histone H2A.X (Ser139) Antibody.

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded L36 pancreatic adenocarcinoma xenografts, untreated (left) or chemotherapy treated (right). (High magnification inset) (Tissue provided by Dr. Murray Resnick, Rhode Island Hospital).

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human breast tumor control (left) or lambda phosphatase-treated (right) using Phospho-Histone H2A.X (Ser139) Antibody.

Flow Cytometry

Flow cytometric analysis of HeLa cells, untreated (blue) and UV-treated (green), using Phospho-Histone H2A.X (Ser139) Antibody compared with a nonspecific negative control antibody (red).

IF-IC

Confocal microscopic images of HeLa cells, UV treated (A) and untreated (B), showing nuclear stain with Phospho-Histone H2A.X (Ser139) Antibody (red) and Phospho-SAPK/JNK (Thr183/Tyr185) (G9) Mouse mAb #9255 (green).

Histone H2A.X is a variant histone that represents approximately 10% of the total H2A histone proteins in normal human fibroblasts (1). H2A.X is required for checkpoint-mediated cell cycle arrest and DNA repair following double-stranded DNA breaks (1). DNA damage, caused by ionizing radiation, UV-light, or radiomimetic agents, results in rapid phosphorylation of H2A.X at Ser139 by PI3K-like kinases, including ATM, ATR, and DNA-PK (2,3). Within minutes following DNA damage, H2A.X is phosphorylated at Ser139 at sites of DNA damage (4). This very early event in the DNA-damage response is required for recruitment of a multitude of DNA-damage response proteins, including MDC1, NBS1, RAD50, MRE11, 53BP1, and BRCA1 (1). In addition to its role in DNA-damage repair, H2A.X is required for DNA fragmentation during apoptosis and is phosphorylated by various kinases in response to apoptotic signals. H2A.X is phosphorylated at Ser139 by DNA-PK in response to cell death receptor activation, c-Jun N-terminal Kinase (JNK1) in response to UV-A irradiation, and p38 MAPK in response to serum starvation (5-8). H2A.X is constitutively phosphorylated on Tyr142 in undamaged cells by WSTF (Williams-Beuren syndrome transcription factor) (9,10). Upon DNA damage, and concurrent with phosphorylation of Ser139, Tyr142 is dephosphorylated at sites of DNA damage by recruited EYA1 and EYA3 phosphatases (9). While phosphorylation at Ser139 facilitates the recruitment of DNA repair proteins and apoptotic proteins to sites of DNA damage, phosphorylation at Tyr142 appears to determine which set of proteins are recruited. Phosphorylation of H2A.X at Tyr142 inhibits the recruitment of DNA repair proteins and promotes binding of pro-apoptotic factors such as JNK1 (9). Mouse embryonic fibroblasts expressing only mutant H2A.X Y142F, which favors recruitment of DNA repair proteins over apoptotic proteins, show a reduced apoptotic response to ionizing radiation (9). Thus, it appears that the balance of H2A.X Tyr142 phosphorylation and dephosphorylation provides a switch mechanism to determine cell fate after DNA damage.

1. Yuan, J. et al. (2010) FEBS Lett 584, 3717-24.
2. Rogakou, E.P. et al. (1998) J Biol Chem 273, 5858-68.
3. Burma, S. et al. (2001) J Biol Chem 276, 42462-7.
4. Rogakou, E.P. et al. (1999) J Cell Biol 146, 905-16.
5. Mukherjee, B. et al. (2006) DNA Repair (Amst) 5, 575-90.
6. Solier, S. et al. (2009) Mol Cell Biol 29, 68-82.
7. Lu, C. et al. (2006) Mol Cell 23, 121-32.
8. Lu, C. et al. (2008) FEBS Lett 582, 2703-8.
9. Cook, P.J. et al. (2009) Nature 458, 591-6.
10. Xiao, A. et al. (2009) Nature 457, 57-62.

• Tran, J.C. et al. (2011) Nature , . Applications: IF-IC (In Cells) Western Blotting

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