Product Pathways - Akt Signaling
SignalSilence® GSK-3α/β siRNA Kit #6300
Kit Includes
- SignalSilence®GSK-3alpha/beta siRNA #6301
- pre-validated single sequence siRNA duplex guaranteed to specifically inhibit GSK-3alpha and beta 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 GSK-3beta (27C10) Rabbit mAb #9315
- to confirm silencing of GSK-3beta expression.
- Non-targeted Bcl-xL Antibody #2762
- to control for protein loading and GSK-3alpha/beta 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 blot analysis of extracts from HeLa cells, untransfected or transfected with GSK-3alpha/beta siRNA. GSK-3beta was detected using a total GSK-3beta antibody, and Bcl-xL was detected using Bcl-xL Antibody #2762. The GSK-3beta Antibody confirms silencing of GSK-3beta expression, and the Bcl-xL Antibody is used to control for loading and siRNA specificity.
Western Blotting
Western blot analysis of extracts from HeLa cells, untransfected or transfected with either GSK-3alpha/beta siRNA or GSK-3alpha siRNA. GSK-3alpha and GSK-3beta were detected using a GSK-3alpha/beta antibody, and eIF4B was detected using eIF4B Antibody #3592. The GSK-3alpha/beta antibody confirms silencing of GSK-3alpha and beta expression, and the eIF4B Antibody is used to control for loading and siRNA specificity.
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® GSK-3?/? siRNA Kit from Cell Signaling Technology allows the researcher to specifically inhibit GSK-3?/? 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® GSK-3alpha/beta siRNA Kit includes GSK-3?/? siRNA, a target-specific GSK-3? Antibody to confirm the silencing of GSK-3? expression and a non-target antibody (Bcl-xL) to control for loading and monitor specificity of GSK-3?/? 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 GSK-3α/β siRNA 48 hours prior to cell lysis. See Protocol for transfection procedure.
Background
Glycogen synthase kinase-3 (GSK-3) was initially identified as an enzyme that regulated glycogen synthesis in response to insulin (1). GSK-3 is a ubiquitously expressed serine/threonine protein kinase that phosphorylates and inactivates glycogen synthase. GSK-3 is a critical downstream element of the PI3 kinase/Akt cell survival pathway, and its activity can be inhibited by Akt-mediated phosphorylation at Ser21 of GSK-3α and Ser9 of GSK-3β (2,3). GSK-3 has been implicated in the regulation of cell fate in Dictyostelium, and is a component of the Wnt signaling pathway required for Drosophila, Xenopus and mammalian development (4). GSK-3 has been shown to regulate cyclin D1 proteolysis and subcellular localization (5).
Small Interfering RNA (siRNA) has been used to specifically silence GSK-3α and β expression, causing an upregulation of β-catenin protein levels (6).
- Welsh, G.I. et al. (1996) Trends Cell. Biol. 6, 274-279.
- Srivastava, A.K. and Pandey, S.K. (1998) Mol. Cell. Biochem. 182, 135-141.
- Cross, D.A. et al. (1995) Nature 378, 785-789.
- Nusse, R. (1997) Cell 89, 321-323.
- Diehl, J.A. et al. (1998) Genes Dev. 12, 3499-3511.
- Yu, J. Y. et al. (2003) Mol. Ther. 7, 228-236.
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!
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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.
