Product Pathways - Translational Control
SignalSilence® p70/85 S6 Kinase siRNA II #6572
Species cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.
Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-), SignalSilence® p70/85 S6 Kinase siRNA I #6566 (+) or SignalSilence® p70/85 S6 Kinase siRNA II (+), using p70 S6 Kinase (49D7) Rabbit mAb #2708 and α-Tubulin (11H10) Rabbit mAb #2125. The p70 S6 Kinase (49D7) Rabbit mAb confirms silencing of p70/85 S6 kinase expression, while the α-Tubulin (11H10) Rabbit mAb is used to control for loading and specificity of p70/85 S6 kinase siRNA.
SignalSilence® p70/85 S6 Kinase siRNA II from Cell Signaling Technology (CST) allows the researcher to specifically inhibit p70/85 S6 kinase expression using RNA interference, a method whereby gene expression can be selectively silenced through the delivery of double stranded RNA molecules into the cell. All SignalSilence® siRNA products are rigorously tested in-house and have been shown to reduce target protein expression by western analysis.
Oligonucleotide synthesis is monitored base by base through trityl analysis to ensure appropriate coupling efficiency. The oligo is subsequently purified by affinity-solid phase extraction. The annealed RNA duplex is further analyzed by mass spectrometry to verify the exact composition of the duplex. Each lot is compared to the previous lot by mass spectrometry to ensure maximum lot-to-lot consistency.
Directions for Use
CST recommends transfection with 100 nM p70/85 S6 Kinase siRNA II 48 to 72 hours prior to cell lysis. For transfection procedure, follow protocol provided by the transfection reagent manufacturer. Please feel free to contact CST with any questions on use.
Each vial contains the equivalent of 100 transfections, which corresponds to a final siRNA concentration of 100 nM per transfection in a 24-well plate with a total volume of 300 μl per well.
p70 S6 kinase is a mitogen activated Ser/Thr protein kinase that is required for cell growth and G1 cell cycle progression (1,2). p70 S6 kinase phosphorylates the S6 protein of the 40S ribosomal subunit and is involved in translational control of 5' oligopyrimidine tract mRNAs (1). A second isoform, p85 S6 kinase, is derived from the same gene and is identical to p70 S6 kinase except for 23 extra residues at the amino terminus, which encode a nuclear localizing signal (1). Both isoforms lie on a mitogen activated signaling pathway downstream of phosphoinositide-3 kinase (PI-3K) and the target of rapamycin, FRAP/mTOR, a pathway distinct from the Ras/MAP kinase cascade (1). The activity of p70 S6 kinase is controlled by multiple phosphorylation events located within the catalytic, linker and pseudosubstrate domains (1). Phosphorylation of Thr229 in the catalytic domain and Thr389 in the linker domain are most critical for kinase function (1). Phosphorylation of Thr389, however, most closely correlates with p70 kinase activity in vivo (3). Prior phosphorylation of Thr389 is required for the action of phosphoinositide 3-dependent protein kinase 1 (PDK1) on Thr229 (4,5). Phosphorylation of this site is stimulated by growth factors such as insulin, EGF and FGF, as well as by serum and some G-protein-coupled receptor ligands, and is blocked by wortmannin, LY294002 (PI-3K inhibitor) and rapamycin (FRAP/mTOR inhibitor) (1,6,7). Ser411, Thr421 and Ser424 lie within a Ser-Pro-rich region located in the pseudosubstrate region (1). Phosphorylation at these sites is thought to activate p70 S6 kinase via relief of pseudosubstrate suppression (1,2). Another LY294002 and rapamycin sensitive phosphorylation site, Ser371, is an in vitro substrate for mTOR and correlates well with the activity of a partially rapamycin resistant mutant p70 S6 kinase (8).
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- Dufner, A. and Thomas, G. (1999) Exp. Cell Res. 253, 100-109.
- Weng, Q.P. et al. (1998) J. Biol. Chem. 273, 16621-16629.
- Pullen, N. et al. (1998) Science 279, 707-710.
- Alessi, D.R. et al. (1998) Curr. Biol. 8, 69-81.
- Polakiewicz, R.D. et al. (1998) J. Biol. Chem. 273, 23534-23541.
- Fingar, D.C. et al. (2002) Genes Dev. 16, 1472-1487.
- Saitoh, M. et al. (2002) J. Biol. Chem. 277, 20104-20112.
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- 6201 SignalSilence® Control siRNA (Fluorescein Conjugate)
- 6568 SignalSilence® Control siRNA (Unconjugated)
- 6566 SignalSilence® p70/85 S6 Kinase siRNA I
- 2708 p70 S6 Kinase (49D7) Rabbit mAb
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.
For Research Use Only. Not For Use In Diagnostic Procedures.