Cell Signaling Technology

Product Pathways - Cell Cycle / Checkpoint

SignalSilence® TTK siRNA II #6368

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 OVCAR8 cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-), SignalSilence® TTK siRNA I #6367 (+) or SignalSilence® TTK siRNA II (+), using TTK (D15B7) Rabbit mAb #5469 (upper) or α-Tubulin (11H10) Rabbit mAb #2125 (lower). The TTK (D15B7) Rabbit mAb confirms silencing of TTK expression, while the α-Tubulin (11H10) Rabbit mAb is used as a loading control.

Description

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

TTK (Mps1, PYT) is a cell cycle regulated dual specificity kinase present in rapidly proliferating tissues and cell lines (1-3). TTK localizes to kinetochores and centromeres and is an essential component of the mitotic spindle checkpoint as well as centrosome duplication (4-6). The mitotic checkpoint inhibits entry into anaphase until all chromosomes are attached to the spindle; inhibition of this process leads to genomic instability and tumorigenesis. Phosphorylation of the BLM helicase at Ser144 by TTK maintains chromosome stability during mitosis (7). Small molecule inhibitors of TTK can block the spindle checkpoint response, thereby making TTK a potential therapeutic target (8,9).TTK also participates in the DNA damage response by directly phosphorylating and activating the cell cycle checkpoint kinase Chk2 at Thr68. Two targets phosphorylated by Chk2 are the cell cycle phosphatase cdc25 and the transcription factor p53. Inactivation of cdc25 phosphatase results in the accumulation of inactive cyclin B and cell cycle arrest following DNA damage. Phosphorylation of p53 by active Chk2 stabilizes the transcription factor and promotes cell cycle arrest and apoptosis in response to DNA damage (10).

  1. Mills, G.B. et al. (1992) J. Biol. Chem. 267, 16000-16006.
  2. Stucke, V.M. et al. (2002) EMBO J. 21, 1723-1732.
  3. Lindberg, R.A. et al. (1993) Oncogene 8, 351-359.
  4. Fisk, H.A. et al. (2003) Proc. Natl. Acad. Sci. USA 100, 14875-14880.
  5. Dou, Z. et al. (2003) Cell Res. 13, 443-449.
  6. Abrieu, A. et al. (2001) Cell 106, 83-93.
  7. Leng, M. et al. (2006) Proc. Natl. Acad. Sci. USA 103, 11485-11490.
  8. Schmidt, M. et al. (2005) EMBO Rep. 6, 866-872.
  9. Dorer, R.K. et al. (2005) Curr. Biol. 15, 1070-1076.
  10. Wei, J.H. et al. (2005) J. Biol. Chem. 280, 7748-7757.

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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