Cell Signaling Technology

Product Pathways - Chromatin Regulation / Epigenetics

SignalSilence® EWS siRNA II #12216

Applications Reactivity
Transfection H (M) (R) (Mk)

Reactivity Key:  H=Human  M=Mouse  R=Rat  Mk=Monkey
Species cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Western Blotting

Western Blotting

Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-), SignalSilence® EWS siRNA I #12175 (+), or SignalSilence® EWS siRNA II (+), using EWS Antibody #11910 (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). The EWS Antibody confirms silencing of EWS expression, while the β-Actin (D6A8) Rabbit mAb is used as a loading control.

Description

SignalSilence® EWS siRNA II from Cell Signaling Technology (CST) allows the researcher to specifically inhibit EWS 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 from CST are rigorously tested in-house and have been shown to reduce target protein expression by western analysis.

Quality Control

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 SignalSilence® EWS 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.

Background

The Ewing sarcoma (EWS) protein is a member of the multifunctional FET (FUS, EWS, and TAF15) family of proteins (1,2). These proteins are RNA and DNA binding proteins that are thought to be important for both transcriptional regulation and RNA processing. EWS can be found as part of a fusion protein with various E-twenty six (ETS) family transcription factors, most commonly Fli-1, in the Ewing sarcoma family of tumors (1-4). The amino terminus of the EWS protein, containing the transcriptional activation domain, is fused to the DNA binding domain of the ETS transcription factor, causing aberrant expression of target genes (1-5). EWS interacts with the transcription initiation complex via TFIID and RNA polymerase II subunits, as well as transcriptional regulators, such as Brn3A and CBP/p300, which suggests a role for EWS in transcriptional regulation (1,6-9). EWS also interacts with multiple components of the splicing machinery, implicating a role for EWS in RNA processing (1,10-12). EWS regulates the expression of cyclin D1, which controls G1-S phase transition during the cell cycle, at the level of transcriptional activation and mRNA splicing. The EWS-Fli-1 fusion protein has been shown to promote the expression of the cyclin D1b splice variant in Ewing sarcoma cells (13). In addition, EWS regulates the DNA damage-induced alternative splicing of genes involved in DNA repair and stress response and is required for cell viability upon DNA damage (14). Consistent with these results, EWS knockout mice display hypersensitivity to ionizing radiation and premature cellular senescence, suggesting a role for EWS in homologous recombination and maintenance of genomic stability (15).

  1. Law, W.J. et al. (2006) Brief Funct Genomic Proteomic 5, 8-14.
  2. Kovar, H. (2011) Sarcoma 2011, 837474.
  3. Delattre, O. et al. (1992) Nature 359, 162-5.
  4. May, W.A. et al. (1993) Mol Cell Biol 13, 7393-8.
  5. Sorensen, P.H. et al. (1994) Nat Genet 6, 146-51.
  6. Bertolotti, A. et al. (1996) EMBO J 15, 5022-31.
  7. Bertolotti, A. et al. (1998) Mol Cell Biol 18, 1489-97.
  8. Araya, N. et al. (2003) J Biol Chem 278, 5427-32.
  9. Thomas, G.R. and Latchman, D.S. Cancer Biol Ther 1, 428-32.
  10. Chansky, H.A. et al. (2001) Cancer Res 61, 3586-90.
  11. Yang, L. et al. (2000) J Biol Chem 275, 37612-8.
  12. Knoop, L.L. and Baker, S.J. (2001) J Biol Chem 276, 22317-22.
  13. Sanchez, G. et al. (2008) Proc Natl Acad Sci U S A 105, 6004-9.
  14. Paronetto, M.P. et al. (2011) Mol Cell 43, 353-68.
  15. Li, H. et al. (2007) J Clin Invest 117, 1314-23.

Application References

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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.

For Research Use Only. Not For Use In Diagnostic Procedures.

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