Cell Signaling Technology

Product Pathways - Translational Control

SignalSilence® mTOR siRNA Kit #6380

Kit Includes

SignalSilence® mTOR siRNA #6381
pre-validated single sequence siRNA duplex guaranteed to specifically inhibit mTOR 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.Kit contains enough for 4(S) or 10 (L) transfections.
Targeted mTOR Antibody #2972
to confirm silencing of mTOR expression.
Non-targeted eIF4B Antibody #3592
to control for protein loading and mTOR 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 either control siRNA (-) or mTOR siRNA (+). mTOR was detected using mTOR Antibody #2972, and eIF4B was detected using eIF4B Antibody #3592. The mTOR Antibody confirms silencing of mTOR expression, and the eIF4B Antibody is used to control for loading and siRNA specificity.

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® mTOR siRNA Kit from Cell Signaling Technology allows the researcher to specifically inhibit mTOR 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 in specified cell lines. SignalSilence® mTOR siRNA Kit includes mTOR siRNA, a target-specific mTOR Antibody to confirm the silencing of mTOR expression and a non-target antibody (eIF4B) to control for loading and monitor specificity of mTOR 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 mTOR siRNA 48 hours prior to cell lysis. See protocol for transfection procedure.

Tested Cell Lines

HeLa

Background

The mammalian target of rapamycin (mTOR, FRAP, RAFT) is a Ser/Thr protein kinase (1-3) that functions as an ATP and amino acid sensor to balance nutrient availability and cell growth (4,5). When sufficient nutrients are available, mTOR responds to a phosphatidic acid-mediated signal to transmit a positive signal to p70 S6 kinase and participate in the inactivation of the eIF4E inhibitor, 4E-BP1 (6). These events result in the translation of specific mRNA subpopulations. mTOR is phosphorylated at Ser2448 via the PI3 kinase/Akt signaling pathway and autophosphorylated at Ser2481 (7,8). mTOR plays a key role in cell growth and homeostasis and may be abnormally regulated in tumors. For these reasons, mTOR is currently under investigation as a potential target for anti-cancer therapy (9).

siRNA has been used to silence mTOR expression in 293-HEK cells, causing decreased raptor expression, decreased p70 S6 kinase phosphorylation, and reduced cell size (9).

  1. Sabers, C.J. et al. (1995) J. Biol. Chem. 270, 815-822.
  2. Brown, E.J. et al. (1994) Nature 369, 756-758.
  3. Sabatini, D.M. et al. (1994) Cell 78, 35-43.
  4. Gingras, A.C. et al. (2001) Genes Dev. 15, 807-826.
  5. Dennis, P.B. et al. (2001) Science 294, 1102-1105.
  6. Fang, Y. et al. (2001) Science 294, 1942-1945.
  7. Nave, B.T. et al. (1999) Biochem. J. 344 Pt 2, 427-431.
  8. Peterson, R.T. et al. (2000) J. Biol. Chem. 275, 7416-7423.
  9. Huang, S. and Houghton, P.J. (2003) Curr. Opin. Pharmacol. 3, 371-377.
  10. Kim, D. H. et al. (2002) Cell 110, 163-175.

Application References

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

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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