Cell Signaling Technology

Product Pathways - Development

SignalSilence® LRP6 siRNA II #8623

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 293 cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-), SignalSilence® LRP6 siRNA I #8650 (+) or SignalSilence® LRP6 siRNA II (+), using LRP6 (C5C7) Rabbit mAb #2560 (upper) or α-Tubulin (11H10) Rabbit mAb #2125 (lower). The LRP6 (C5C7) Rabbit mAb confirms silencing of LRP6 expression, while the α-Tubulin (11H10) Rabbit mAb is used as a loading control.

Description

SignalSilence® LRP6 siRNA II from Cell Signaling Technology (CST) allows the researcher to specifically inhibit LRP6 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® LRP6 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

LRP5 and LRP6 are single-pass transmembrane proteins belonging to the low-density lipoprotein receptor (LDLR)-related protein family. Unlike other members of the LDLR family, LRP5 and LRP6 have four EGF and three LDLR repeats in the extracellular domain, and proline-rich motifs in the cytoplasmic domain (1). They function as co-receptors for Wnt and are required for the canonical Wnt/β-catenin signaling pathway (2,3). LRP5 and LRP6 are highly homologous and have redundant roles during development (4,5). The activity of LRP5 and LRP6 can be inhibited by the binding of some members of the Dickkopf (DKK) family of proteins (6,7). Upon stimulation with Wnt, LRP6 is phosphorylated at multiple sites including Thr1479, Ser1490, and Thr1493 by kinases such as GSK-3 and CK1 (8-10). Phosphorylated LRP6 recruits axin to the membrane and presumably activates β-catenin signaling (8-10).

  1. Brown, S.D. et al. (1998) Biochem. Biophys. Res. Commun. 248, 879-888.
  2. Pinson, K.I. et al. (2000) Nature 407, 535-538.
  3. Tamai, K. et al. (2000) Nature 407, 530-535.
  4. Kelly, O.G. et al. (2004) Development 131, 2803-2815.
  5. He, X. et al. (2004) Development 131, 1663-1677.
  6. Semënov, M.V. et al. (2001) Curr Biol 11, 951-61.
  7. Bafico, A. et al. (2001) Nat. Cell Biol. 3, 683-668.
  8. Tamai, K. et al. (2004) Mol. Cell 13, 149-156.
  9. Zeng, X. et al. (2005) Nature 438, 873-877.
  10. Davidson, G. et al. (2005) Nature 438, 867-872.

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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