Cell Signaling Technology

Product Pathways - Cell Cycle / Checkpoint

SignalSilence® p53 siRNA Kit (Human Specific) #6230

Kit Includes

SignalSilence® p53 siRNA (Human Specific) #6231
pre-validated single sequence siRNA duplex guaranteed to specifically inhibit p53 expression in specified cell lines.
SignalSilence® Control siRNA (Fluorescein Conjugate) #6201
a non-targeted negative control duplex designed to monitor transfection efficiency using microscopy and to monitor siRNA specificity.
Targeted p53 Antibody #9282
to confirm silencing of p53 expression.
Non-targeted p42 MAPK Antibody #9108
to control for protein loading and p53 siRNA specificity.
*Transfection Reagent
designed specifically for highly efficient siRNA delivery in mammalian cells. Transfection reagent is supplied by Mirus.

*Store at 4°C (packaged separately)

Western Blotting

Western Blotting

Western blot analysis of extracts from HeLa cells transfected with non-targeted (-) or targeted (+) siRNA. p53 was detected using the p53 Antibody #9282, and p42 MAPK was detected using the p42 MAPK Antibody #9108. The p53 Antibody confirms silencing of p53 expression, and the p42 MAPK Antibody was used to control for loading and specificity of p53 siRNA.

Fluorescent Detection

Fluorescent Detection

Fluorescent detection of SignalSilence® Control siRNA (Fluorescein Conjugate) #6201 in living HeLa cells 24 hours post-transfection, demonstrating nearly 100% transfection efficiency.

Description

SignalSilence® p53 siRNA Kit from Cell Signaling Technology allows the researcher to specifically inhibit p53 expression. The kit utilizes RNA interference, a method in which gene expression can be selectivelysilenced through the delivery of double stranded RNA molecules into the cell. All SignalSilence® siRNA kits are rigorously tested in-house and have been shown to reduce protein expression. SignalSilence® p53 siRNA Kit includes p53 siRNA, a target-specific p53 Antibody to confirm the silencing of p53 expression and a non-target antibody (p42 MAPK) to control for loading and monitor specificity of p53 siRNA. Additionally, a fluorescein-labeled non-targeted siRNA control allows the user to monitor transfection efficiency and siRNA specificity. The kit also contains a transfection reagent designed solely for efficient siRNA delivery in mammalian cells.

Directions for Use

CST recommends transfection with 50 nM human-specific p53 siRNA 24 hours prior to cell lysis. See protocol for transfection procedure.

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 (6,7). 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 (4,5). Chk2 and Chk1 can phosphorylate p53 at Ser20, enhancing its tetramerization, stability and activity (8,9). p53 is phosphorylated at Ser392 in vivo (11,12) and by CAK in vitro (12). Phosphorylation of p53 at Ser392 is increased in human tumors (14) and has been reported to influence the growth suppressor function, DNA binding and transcriptional activation of p53 (10,11,13). p53 is phosphorylated at Ser6 and Ser9 by CK1δ and CK1ε both in vitro and in vivo (10,15). 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).

Small interfering RNA (siRNA) has been used to specifically silence p53 expression in human tumor cells (17).

  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. Tibbetts, R.S. et al. (1999) Genes Dev. 13, 152-157.
  6. Chehab, N.H. et al. (1999) Proc. Natl. Acad. Sci. USA 96, 13777-13782.
  7. Honda, R. et al. (1997) FEBS Lett. 420, 25-27.
  8. Shieh, S.Y. et al. (1999) EMBO J. 18, 1815-1823.
  9. Hirao, A. et al. (2000) Science 287, 1824-1827.
  10. Kohn, K.W. (1999) Mol. Biol. Cell 10, 2703-2734.
  11. Hao, M. et al. (1996) J. Biol. Chem. 271, 29380-29385.
  12. Lu, H. et al. (1997) Mol. Cell. Biol. 17, 5923-5934.
  13. Lohrum, M. and Scheidtmann, K.H. (1996) Oncogene 13, 2527-2539.
  14. Ulrich, S.J. et al. (1993) Proc. Natl. Acad. Sci. USA 90, 5954-5958.
  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.
  20. Irwin, M. S. et al. (2003) Cancer Cell 3(4) , 403-410.

Application References

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Companion Products

Limited Use Label License, RNA interference: This product is licensed under European Patent 1144623 and foreign equivalents from Ribopharma AG, Kulmbach, Germany and is provided only for use in non-commercial research specifically excluding use (a) in drug discovery or drug development, including target identification or target validation, by or on behalf of a commercial entity, (b) for contract research or commercial screening services, (c) for the production or manufacture of siRNA-related products for sale, or (d) for the generation of commercial databases for sale to Third Parties. Information about licenses for these and other commercial uses is available from Ribopharma AG, Fritz-Hornschuch-Str. 9, D-95326 Kulmbach, Germany.

This product is for in vitro research use only and is not intended for use in humans or animals. This product is not intended for use as therapeutic or in diagnostic procedures.

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