Product Pathways - Akt Signaling

SignalSilence® Akt siRNA Kit #6210

• pathway
• more info
• application references
• datasheet PDF
• MSDS PDF
• protocols

Kit Includes

SignalSilence^® Akt siRNA #6211

pre-validated single sequence siRNA duplex guaranteed to specifically inhibit Akt 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 Akt Antibody #9272

to confirm silencing of Akt expression.

Non-targeted p42 MAPK Antibody #9108

to control for protein loading and Akt 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)

Description

SignalSilence® Akt siRNA Kit from Cell Signaling Technology allows the researcher to specifically inhibit Akt 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® Akt siRNA Kit includes Akt siRNA, a target-specific Akt Antibody to confirm the silencing of Akt expression and a non-target antibody (p42 MAPK) to control for loading and monitor specificity of Akt 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 SignalSilence® Akt siRNA 48 hours prior to cell lysis. See protocol for transfection procedure.

Background

Akt, also referred to as PKB or Rac, plays a critical role in controlling survival and apoptosis (1-3). This protein kinase is activated by insulin and various growth and survival factors to function in a wortmannin-sensitive pathway involving PI3 kinase (2,3). Akt is activated by phospholipid binding and activation loop phosphorylation at Thr308 by PDK1 (4) and by phosphorylation within the carboxy terminus at Ser473. The previously elusive PDK2 responsible for phosphorylation of Akt at Ser473 has been identified as mammalian target of rapamycin (mTor) in a rapamycin-insensitive complex with rictor and Sin1 (5,6). Akt promotes cell survival by inhibiting apoptosis by phosphorylating and inactivating several targets, including Bad (7), forkhead transcription factors (8), c-Raf (9) and caspase-9. PTEN phosphatase is a major negative regulator of the PI3 kinase/Akt signaling pathway (10). LY294002 is a specific PI3 kinase inhibitor (11).Another essential Akt function is the regulation of glycogen synthesis through phosphorylation and inactivation of GSK-3α and β (12,13). Akt may also play a role in insulin stimulation of glucose transport (12).In addition to its role in survival and glycogen synthesis, Akt is involved in cell cycle regulation by preventing GSK-3β mediated phosphorylation and degradation of cyclin D1 (14) and by negatively regulating the cyclin dependent kinase inhibitors p27 Kip (15) and p21 Waf1/CIP1 (16). Akt also plays a critical role in cell growth by directly phosphorylating mTOR in a rapamycin-sensitive complex containing raptor (17). More importantly, Akt phosphorylates and inactivates tuberin (TSC2), an inhibitor of mTOR within the mTOR-raptor complex (18). Inhibition of mTOR stops the protein synthesis machinery due to inactivation of its effector, p70 S6 kinase and activation of the eukaryotic initiation factor 4E binding protein 1 (4E-EP1), an inhibitor of translation (18,19).

Small interfering RNA (siRNA) has been used to specifically silence Akt in CHO cells and 3T3-L1 adipocytes (18)

1. Franke, T.F. et al. (1997) Cell 88, 435-7.
2. Burgering, B.M. and Coffer, P.J. (1995) Nature 376, 599-602.
3. Franke, T.F. et al. (1995) Cell 81, 727-36.
4. Alessi, D.R. et al. (1996) EMBO J 15, 6541-51.
5. Sarbassov, D.D. et al. (2005) Science 307, 1098-101.
6. Jacinto, E. et al. (2006) Cell 127, 125-37.
7. Cardone, M.H. et al. (1998) Science 282, 1318-21.
8. Brunet, A. et al. (1999) Cell 96, 857-68.
9. Zimmermann, S. and Moelling, K. (1999) Science 286, 1741-4.
10. Cantley, L.C. and Neel, B.G. (1999) Proc Natl Acad Sci USA 96, 4240-5.
11. Vlahos, C.J. et al. (1994) J Biol Chem 269, 5241-8.
12. Hajduch, E. et al. (2001) FEBS Lett 492, 199-203.
13. Cross, D.A. et al. (1995) Nature 378, 785-9.
14. Diehl, J.A. et al. (1998) Genes Dev 12, 3499-511.
15. Gesbert, F. et al. (2000) J Biol Chem 275, 39223-30.
16. Zhou, B.P. et al. (2001) Nat Cell Biol 3, 245-52.
17. Nave, B.T. et al. (1999) Biochem J 344 Pt 2, 427-31.
18. Inoki, K. et al. (2002) Nat Cell Biol 4, 648-57.
19. Manning, B.D. et al. (2002) Mol Cell 10, 151-62.
20. Katome, T. et al. (2003) J. Biol. Chem. 278(30) , 28312-28323.

Application References

• Kakazu, A. et al. (2004) Invest Ophthalmol Vis Sci 45, 3485-92. This article references the use of SignalSilence® Akt siRNA Kit in the following applications: Western Blotting siRNA
• Kierbel, A. et al. (2005) Mol Biol Cell 16, 2577-85. This article references the use of SignalSilence® Akt siRNA Kit in the following applications: Western Blotting siRNA
• Wu, K.L. et al. (2004) J Biol Chem 279, 26280-6. This article references the use of SignalSilence® Akt siRNA Kit in the following applications: Western Blotting siRNA
• Chandrasekar, B. et al. (2005) J Biol Chem 280, 4553-67. This article references the use of SignalSilence® Akt siRNA Kit in the following applications: Western Blotting siRNA

Have you published research involving the use of our products? If so we'd love to hear about it. Please let us know!

Companion Products

• 6201 SignalSilence® Control siRNA (Fluorescein Conjugate)
• 7074 Anti-rabbit IgG, HRP-linked Antibody
• 7720 Prestained Protein Marker, Broad Range (Premixed Format)
• 7727 Biotinylated Protein Ladder Detection Pack
• 7003 20X LumiGLO^® Reagent and 20X Peroxide

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.