Cell Signaling Technology

Product Pathways - Akt Signaling

SignalSilence® PTEN siRNA Kit (Human Specific) #6250

Kit Includes

SignalSilence® PTEN siRNA (Human Specific) #6251
pre-validated single sequence siRNA duplex guaranteed to specifically inhibit PTEN 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 PTEN Antibody #9552
to confirm silencing of PTEN expression.
Non-targeted p42 MAPK Antibody #9108
to control for protein loading and PTEN 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. PTEN wasdetected using the PTEN Antibody #9552, and p42 was detected using the p42 MAPK Antibody #9108. The PTEN Antibody confirms silencing of PTEN expression, and the p42 MAPK Antibody is used to control for loading and specificity of PTEN 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® PTEN siRNA Kit from Cell Signaling Technology allows the researcher to specifically inhibit PTEN expression. The kit utilizes RNA interference, a method in which gene expression can be selectively silenced 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® PTEN siRNA Kit includes PTEN siRNA, a target-specific PTEN Antibody to confirm the silencing of PTEN expression and a non-target antibody (p42 MAPK) to control for loading and monitor specificity of PTEN 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 100 nM PTEN siRNA 48 hours prior to cell lysis. See protocol for transfection procedure.

Background

PTEN (phosphatase and tensin homologue deleted on chromosome ten), also referred to as MMAC (mutated in multiple advanced cancers) phosphatase, is a tumor suppressor implicated in a wide variety of human cancers (1). PTEN encodes a 403 amino acid polypeptide originally described as a dual-specificity protein phosphatase (2). The main substrates of PTEN are inositol phospholipids generated by the activation of the phosphoinositide 3-kinase (PI3K) (3). PTEN is a major negative regulator of the PI3K/Akt signaling pathway (1,4,5). PTEN possesses a carboxy-terminal, noncatalytic regulatory domain with three phosphorylation sites (Ser380, Thr382 and Thr383) that regulate PTEN stability and may affect its biological activity (6,7). PTEN regulates p53 protein level and activity (8) and is involved in G protein coupled signaling during chemotaxis (9,10).

Small Interfering RNA (siRNA) has been used to specifically silence PTEN expression in HeLa cells (10).

  1. Cantley, L.C. and Neel, B.G. (1999) Proc. Natl. Acad. Sci. USA 96, 4240-4245.
  2. Myers, M.P. et al. (1997) Proc. Natl. Acad. Sci. USA 94, 9052-9057.
  3. Myers, M.P. et al. (1998) Proc. Natl. Acad. Sci. USA 95, 13513-13518.
  4. Wan, X. and Helman, L.J. (2003) Oncogene 22, 8205-8211.
  5. Wu, X. et al. (1998) Proc. Natl. Acad. Sci. USA 95, 15587-15591.
  6. Vazquez, F. et al. (2000) Mol. Cell. Biol. 20, 5010-5018.
  7. Torres, J. and Pulido, R. (2001) J. Biol. Chem. 276, 993-998.
  8. Freeman, D.J. et al. (2003) Cancer Cell 3, 117-130.
  9. Funamoto, S. et al. (2002) Cell 109, 611-623.
  10. Iijima, M. and Devreotes, P. (2002) Cell 109, 599-610.
  11. Czauderna, F. et al. (2003) Nucleic Acids Res. 31(2) , 670-682.

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.

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