Cell Signaling Technology

Product Pathways - DNA Damage

Phospho-p53 (Ser15) Antibody #9284

Applications Reactivity Sensitivity MW (kDa) Source
W IP IF-IC ChIP H M R Mk Endogenous 53 Rabbit

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IF-IC=Immunofluorescence (Immunocytochemistry)  ChIP=Chromatin IP
Reactivity Key:  H=Human  M=Mouse  R=Rat  Mk=Monkey
Species cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Protocols

Specificity / Sensitivity

Phospho-p53 (Ser15) Antibody detects endogenous levels of p53 only when phosphorylated at serine 15. The antibody does not cross-react with p53 phosphorylated at other sites.

Source / Purification

Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser15 of human p53. Antibodies are purified by protein A and peptide affinity chromatography.

Western Blotting

Western Blotting

Western blot analysis of extracts from MvILu cells treated with UV or hydroxyurea (20 mM) for the indicated times, using Phospho-p53 (Ser15) Antibody.

Western Blotting

Western Blotting

Western blot analysis of a p53 fusion protein, untreated or phosphorylated by DNA-PK, using Phospho-p53 (Ser15) Antibody (upper) and p53 Antibody #9282 (lower).

Western Blotting

Western Blotting

Western blot analysis of extracts from PC12 and HeLa cells treated with UV for the indicated times, using Phospho-p53 (Ser15) Antibody.


IF-IC

IF-IC

Confocal immunofluorescent analysis of HT-29 (p53++, left) and differentiated THP-1 cells (p53- -, right), UV-treated (top) or untreated (bottom), using Phospho-p53 (Ser15) Antibody (green). Actin filaments were labeled with DY-554 phalloidin (red).

Chromatin IP

Chromatin IP

Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 HCT116 cells treated with UV (100 J/m2 followed by a 3 hour recovery) and either 5 μl of Phospho-p53 (Ser15) Antibody or 2 μl of 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 CDKN1A Promoter Primers #6449, human MDM2 intron 2 primers, 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.

Background

The p53 tumor suppressor protein plays a major role in cellular response to DNA damage and other genomic aberrations. Activation of p53 can lead to either cell cycle arrest and DNA repair or apoptosis (1). p53 is phosphorylated at multiple sites in vivo and by several different protein kinases in vitro (2,3). DNA damage induces phosphorylation of p53 at Ser15 and Ser20 and leads to a reduced interaction between p53 and its negative regulator, the oncoprotein MDM2 (4). MDM2 inhibits p53 accumulation by targeting it for ubiquitination and proteasomal degradation (5,6). p53 can be phosphorylated by ATM, ATR, and DNA-PK at Ser15 and Ser37. Phosphorylation impairs the ability of MDM2 to bind p53, promoting both the accumulation and activation of p53 in response to DNA damage (4,7). Chk2 and Chk1 can phosphorylate p53 at Ser20, enhancing its tetramerization, stability, and activity (8,9). p53 is phosphorylated at Ser392 in vivo (10,11) and by CAK in vitro (11). Phosphorylation of p53 at Ser392 is increased in human tumors (12) and has been reported to influence the growth suppressor function, DNA binding, and transcriptional activation of p53 (10,13,14). p53 is phosphorylated at Ser6 and Ser9 by CK1δ and CK1ε both in vitro and in vivo (13,15). Phosphorylation of p53 at Ser46 regulates the ability of p53 to induce apoptosis (16). Acetylation of p53 is mediated by p300 and CBP acetyltransferases. Inhibition of deacetylation suppressing MDM2 from recruiting HDAC1 complex by p19 (ARF) stabilizes p53. Acetylation appears to play a positive role in the accumulation of p53 protein in stress response (17). Following DNA damage, human p53 becomes acetylated at Lys382 (Lys379 in mouse) in vivo to enhance p53-DNA binding (18). Deacetylation of p53 occurs through interaction with the SIRT1 protein, a deacetylase that may be involved in cellular aging and the DNA damage response (19).

  1. Levine, A.J. (1997) Cell 88, 323-331.
  2. Meek, D.W. (1994) Semin. Cancer Biol. 5, 203-210.
  3. Milczarek, G.J. et al. (1997) Life Sci. 60, 1-11.
  4. Shieh, S.Y. et al. (1997) Cell 91, 325-334.
  5. Chehab, N.H. et al. (1999) Proc. Natl. Acad. Sci. USA 96, 13777-13782.
  6. Honda, R. et al. (1997) FEBS Lett. 420, 25-27.
  7. Tibbetts, R.S. et al. (1999) Genes Dev. 13, 152-157.
  8. Shieh, S.Y. et al. (1999) EMBO J. 18, 1815-1823.
  9. Hirao, A. et al. (2000) Science 287, 1824-1827.
  10. Hao, M. et al. (1996) J. Biol. Chem. 271, 29380-29385.
  11. Lu, H. et al. (1997) Mol. Cell. Biol. 17, 5923-5934.
  12. Ullrich, S.J. et al. (1993) Proc. Natl. Acad. Sci. USA 90, 5954-5958.
  13. Kohn, K.W. (1999) Mol. Biol. Cell 10, 2703-2734.
  14. Lohrum, M. and Scheidtmann, K.H. (1996) Oncogene 13, 2527-2539.
  15. Knippschild, U. et al. (1997) Oncogene 15, 1727-1736.
  16. Oda, K. et al. (2000) Cell 102, 849-862.
  17. Ito, A. et al. (2001) EMBO J. 20, 1331-1340.
  18. Sakaguchi, K. et al. (1998) Genes Dev. 12, 2831-2841.
  19. Solomon, J.M. et al. (2006) Mol. Cell. Biol. 26, 28-38.

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This product is intended for research purposes only. The product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

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