Product Pathways - Translational Control
SignalSilence® eIF4E siRNA Kit (Human Specific) #6310
Kit Includes
- SignalSilence® eIF4E siRNA (Human Specific) #6311
- pre-validated single sequence siRNA duplex guaranteed to specifically inhibit eIF4E 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 eIF4E antibody #9742
- to confirm silencing of eIF4E expression.
- Non-targeted eIF4B #3592
- to control for protein loading and eIF4E 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 either nonspecific control siRNA or eIF4E siRNA. eIF4E was detected using eIF4E Antibody #9742, and eIF4B was detected using eIF4B Antibody #3592. The eIF4E Antibody confirms silencing of eIF4E 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® eIF4E siRNA Kit from Cell Signaling Technology allows the researcher to specifically inhibit eIF4E 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® eIF4E siRNA Kit includes eIF4E siRNA, a target-specific eIF4E Antibody to confirm the silencing of eIF4E expression and a non-target antibody (eIF4B) to control for loading and monitor specificity of eIF4E 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 human-specific eIF4E siRNA 48 hours prior to cell lysis. See Protocol for transfection procedure.
Background
Eukaryotic initiation factor 4E (eIF4E) binds to the mRNA cap structure to mediate the initiation of translation (1,2). eIF4E interacts with eIF4G, a scaffold protein that promotes assembly of eIF4E and eIF4A into the eIF4F complex (2). eIF4B is thought to assist the eIF4F complex in translation initiation. Upon activation by mitogenic and/or stress stimuli mediated by Erk and p38 MAPK, Mnk1 phosphorylates eIF4E at Ser209 in vivo (3,4). Two Erk and p38 MAPK phosphorylation sites in mouse Mnk1 (Thr197 and Thr202) are essential for Mnk1 kinase activity (3). The carboxy-terminal region of eIF4G also contains serum-stimulated phosphorylation sites, including Ser1108, Ser1148 and Ser1192 (5). Phosphorylation at these sites is blocked by the PI3 kinase inhibitor LY294002 and by the FRAP/mTOR inhibitor rapamycin.
siRNA has been used to silence eIF4E expression in malignant cholangiocytes, causing decreased anchorage-independent growth (6).
- Sonenberg, N. et al. (1978) Proc. Natl. Acad. Sci. USA 75, 4843-4847.
- Gingras, A.C. et al. (1999) Annu. Rev. Biochem. 68, 913-963.
- Waskiewicz, A. et al. (1999) Mol. Cell. Biol. 19, 1871-1880.
- Pyronnet, S. et al. (1999) EMBO J. 18, 270-279.
- Raught, B. et al. (2000) EMBO J. 19, 434-444.
- Yamagiwa , Y. et al. (2003) Hepatology 38, 158-166.
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
- 9741 Phospho-eIF4E (Ser209) Antibody
- 2111 Phospho-Mnk1 (Thr197/202) Antibody
- 7074 Anti-rabbit IgG, HRP-linked Antibody
- 7727 Biotinylated Protein Ladder Detection Pack
- 7003 20X LumiGLO® Reagent and 20X Peroxide
- 7720 Prestained Protein Marker, Broad Range (Premixed Format)
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.
