Cell Signaling Technology

Product Pathways - Protein Stability

SignalSilence® USP9X siRNA I #6308

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 (-) or SignalSilence® USP9X siRNA I (+), using USP9X Antibody #5751 (upper) or α-Tubulin (11H10) Rabbit mAb #2125 (lower). The USP9X Antibody confirms silencing of USP9X expression, while theα-Tubulin (11H10) Rabbit mAb is used as a loading control.

Description

SignalSilence® USP9X siRNA I from Cell Signaling Technology (CST) allows the researcher to specifically inhibit USP9X 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 USP9X 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

Protein ubiquitination and deubiquitination is a reversible process catalyzed by ubiquitinating enzymes (UBEs) and deubiquitinating enzymes (DUBs) (1,2). DUBs are categorized into five subfamiles-USP, UCH, OTU, MJD, and JAMM. USP9X/hFAM is an X-linked member of the ubiquitin-specific protease (USP) subfamily of DUBs and possesses a well-conserved catalytic domain with cysteine peptidase activity, which allows for cleavage of ubiquitin and polyubiquitin conjugates. USP9X is the mammalian homolog of the Drosophila fat-facets (faf) gene, which is essential for normal eye development and viability of the early fly embryo (3,4). While USP9X expression is also critical for normal mammalian development (5-7), many of its substrates are only beginning to be elucidated. There is mounting evidence that USP9X functions in the formation of cell-cell contacts during the polarization of epithelial cells, in part through deubiquitination-dependent stabilization of molecules involved in maintaining the integrity of both adherens and tight junctions. Indeed, USP9X has been found to associate with AF-6, the β-catenin-E-cadherin complex, and EFA6 (8-11). Studies have also demonstrated that USP9X is an integral component of the TGF-β/BMP signaling cascade and regulates TGF-β responsiveness by opposing TRIM33-mediated monoubiquitination of SMAD4 (12). Recently, USP9X was shown to be overexpressed in a variety of human cancers, which was linked to enhanced cell survival and chemoresistance through its ability to deubiquitinate and stabilize the Mcl-1 oncoprotein. This evidence supports USP9X as a therapeutic target in the context of human malignancies such as follicular lymphomas and diffuse large B-cell lymphomas (13).

  1. Nijman, S.M. et al. (2005) Cell 123, 773-86.
  2. Nalepa, G. et al. (2006) Nat Rev Drug Discov 5, 596-613.
  3. Huang, Y. et al. (1995) Science 270, 1828-31.
  4. Huang, Y. and Fischer-Vize, J.A. (1996) Development 122, 3207-16.
  5. Pantaleon, M. et al. (2001) Mech Dev 109, 151-60.
  6. Noma, T. et al. (2002) Mech Dev 119 Suppl 1, S91-5.
  7. Xu, J. et al. (2005) J Neurosci Res 80, 47-55.
  8. Taya, S. et al. (1998) J Cell Biol 142, 1053-62.
  9. Taya, S. et al. (1999) Genes Cells 4, 757-67.
  10. Murray, R.Z. et al. (2004) Mol Biol Cell 15, 1591-9.
  11. Théard, D. et al. (2010) EMBO J 29, 1499-509.
  12. Dupont, S. et al. (2009) Cell 136, 123-35.
  13. Schwickart, M. et al. (2010) Nature 463, 103-7.

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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