Cell Signaling Technology

Product Pathways - Development

SignalSilence® Frizzled6 siRNA I #6596

Applications Reactivity
Transfection H

Reactivity Key:  H=Human
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® Frizzled6 siRNA I (+) or SignalSilence® Frizzled6 siRNA II #6597 (+), using Frizzled6 (D16E5) Rabbit mAb #5158 (upper) or α-Tubulin (11H10) Rabbit mAb #2125 (lower). The Frizzled6 (D16E5) Rabbit mAb confirms silencing of Frizzled6 expression, while the α-Tubulin (11H10) Rabbit mAb is used as a loading control.

Description

SignalSilence® Frizzled6 siRNA I from Cell Signaling Technology (CST) allows the researcher to specifically inhibit Frizzled6 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 Frizzled6 siRNA I 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

Frizzled (Fzd) belongs to the seven transmembrane-spanning G-protein-coupled receptor (GPCR) superfamily (1). Fzds have a large extracellular N-terminal region containing a cysteine-rich domain (CRD), which is involved in binding to Wnt proteins (1,2). The intracellular C-terminus binds to the PDZ domain of Dvl proteins, a major signaling component downstream of Fzd (3). Wnt proteins bind to Fzd and the co-receptors LRP5 or LPR6, and activate Wnt/β-catenin pathway through inhibiting phosphorylation of β-catenin by GSK3-β (4,5). In addition to this canonical Wnt/β-catenin pathway, some Wnt proteins can also activate the Fzd/Ca2+ pathway and Fzd/PCP (planar cell polarity) pathway (6,7). The mammalian Fzd subfamily has 10 members (Fzd1 to Fzd10) and they may mediate signaling through different pathways (8). Some Fzds can also bind to other secreted proteins, like Norrin and R-Spondin (9-11).

  1. Schulte, G. and Bryja, V. (2007) Trends Pharmacol Sci 28, 518-25.
  2. Hsieh, J.C. et al. (1999) Proc Natl Acad Sci USA 96, 3546-51.
  3. Wong, H.C. et al. (2003) Mol Cell 12, 1251-60.
  4. Zeng, X. et al. (2005) Nature 438, 873-7.
  5. Davidson, G. et al. (2005) Nature 438, 867-72.
  6. Fanto, M. and McNeill, H. (2004) J Cell Sci 117, 527-33.
  7. Kohn, A.D. and Moon, R.T. Cell Calcium 38, 439-46.
  8. Cadigan, K.M. and Liu, Y.I. (2006) J Cell Sci 119, 395-402.
  9. Xu, Q. et al. (2004) Cell 116, 883-95.
  10. Nam, J.S. et al. (2006) J Biol Chem 281, 13247-57.
  11. Hendrickx, M. and Leyns, L. (2008) Dev Growth Differ 50, 229-43.

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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