Cell Signaling Technology

Product Pathways - Jak/Stat Pathway

SignalSilence® PIAS3 siRNA II #9031

Applications Reactivity
Transfection H (Mk)

Reactivity Key:  H=Human  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 RD cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-), SignalSilence® PIAS3 siRNA I #9073 (+), or SignalSilence® PIAS3 siRNA II (+), using PIAS3 (D5F9) XP® Rabbit mAb #9042 (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). The PIAS3 (D5F9) XP® Rabbit mAb confirms silencing of PIAS3 expression, while the β-Actin (D6A8) Rabbit mAb is used as a loading control.

Description

SignalSilence® PIAS3 siRNA II from Cell Signaling Technology (CST) allows the researcher to specifically inhibit PIAS3 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® PIAS3 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 protein inhibitor of activated Stat (PIAS) proteins, which include PIAS1, PIAS3, PIASx, and PIASy, were originally characterized based on their interaction with the Stat family of transcription factors (1,2). PIAS1, PIAS3, and PIASx interact with and repress Stat1, Stat3, and Stat4, respectively (1-3). Deletion of PIAS1 leads to inhibition of interferon-inducible genes and increased protection against infection (4). The PIAS family contains a conserved RING domain that has been linked to a function as a small ubiquitin-related modifier (SUMO) ligase, coupling the SUMO conjugating enzyme Ubc9 with its substrate proteins (5,6). Numerous studies have now shown that PIAS family members can regulate the activity of transcription factors through distinct mechanisms, including NF-κB (7,8), c-Jun, p53 (5,9), Oct-4 (10), and Smads (11,12). The activity of PIAS1 is regulated by both phosphorylation and arginine methylation. Inflammatory stimuli can induce IKK-mediated phosphorylation of PIAS1 at Ser90, which is required for its activity (13). In addition, PRMT1 induces arginine methylation of PIAS1 at Arg303 following interferon treatment and is associated with its repressive activity on Stat1 (14).

  1. Liu, B. et al. (1998) Proc Natl Acad Sci USA 95, 10626-31.
  2. Chung, C.D. et al. (1997) Science 278, 1803-5.
  3. Arora, T. et al. (2003) J Biol Chem 278, 21327-30.
  4. Liu, B. et al. (2004) Nat Immunol 5, 891-8.
  5. Schmidt, D. and Müller, S. (2002) Proc Natl Acad Sci USA 99, 2872-7.
  6. Kotaja, N. et al. (2002) Mol Cell Biol 22, 5222-34.
  7. Liu, B. et al. (2005) Mol Cell Biol 25, 1113-23.
  8. Tahk, S. et al. (2007) Proc Natl Acad Sci USA 104, 11643-8.
  9. Bischof, O. et al. (2006) Mol Cell 22, 783-94.
  10. Tolkunova, E. et al. (2007) J Mol Biol 374, 1200-12.
  11. Long, J. et al. (2004) Proc Natl Acad Sci USA 101, 99-104.
  12. Murdoch, R.N. and Edwards, T. (1992) Biochem Int 28, 1029-37.
  13. Liu, B. et al. (2007) Cell 129, 903-14.
  14. Weber, S. et al. (2009) Genes Dev 23, 118-32.

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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