Cell Signaling Technology

Product Pathways - Apoptosis

SignalSilence® FLIP siRNA I #12372

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 A549 cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-), SignalSilence® FLIP siRNA I (+), or SignalSilence® FLIP siRNA II #12407 (+), using FLIP (D16A8) Rabbit mAb #8510 (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). The FLIP (D16A8) Rabbit mAb confirms silencing of FLIP expression, while the β-Actin (D6A8) Rabbit mAb is used as a loading control.

Description

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

Cellular FLIP (FLICE inhibitory protein) is a regulator of apoptosis that has various names, such as c-FLIP (1), Casper (2), CLARP (3), FLAME (4), I-FLICE (5), MRIT (6), CASH (7), and Usurpin (8). FLIP is expressed as two alternative splice isoforms, FLIP short (FLIPS) and FLIP long (FLIPL). FLIPS contains two death effector domains (DEDs) like those found on the death receptor adaptor protein FADD and the pro-domain of caspase-8. FLIPL shares significant homology with caspase-8 (FLICE), and contains an additional death effector domain, but FLIPL lacks the catalytic active site of the caspases and does not have protease activity. Both FLIP isoforms have been reported to interact with FADD and pro-caspase-8. The role of FLIP in apoptosis is controversial as some research studies have reported it to be anti-apoptotic, while others claim that it is pro-apoptotic. Overexpression of FLIPL can lead to caspase-8 heterodimers that produce an active protease, resulting in apoptosis. However, at physiological levels, it is thought that the binding of FLIP to the DED of FADD results in inhibition of caspase-8 processing. Reduction of FLIP by siRNA or gene targeting sensitizes cells to death receptor-mediated apoptosis. FLIP has also been implicated in the resistance of cancer cells to apoptosis and is upregulated in some cancer types including Hodgkin's lymphoma and ovarian and colon carcinomas (9).

  1. Irmler, M. et al. (1997) Nature 388, 190-195.
  2. Shu, H. B. et al. (1997) Immunity 6, 751-763.
  3. Inohara, N. et al. (1997) Proc. Natl. Acad. Sci. USA 94, 10717-10722.
  4. Srinivasula, S. M. et al. (1997) J. Biol. Chem. 272, 18542-18545.
  5. Hu, S. et al. (1997) J. Biol. Chem. 272, 17255-17257.
  6. Han, D. K. et al. (1997) Proc. Natl. Acad. Sci. USA 94, 11333-11338.
  7. Dita, R. M. et al. (1998) Cell Death Differ. 5, 271-288.
  8. Goltsev, Y. V. et al. (1997) J. Biol. Chem. 272, 19641-19644.
  9. Kataoka, T. (2005) Crit. Rev. Immunol. 25, 31-58.

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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