Cell Signaling Technology

Product Pathways - Cell Cycle / Checkpoint

SignalSilence® PLK1 siRNA I #6292

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 HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-), SignalSilence® PLK1 siRNA I (+), or SignalSilence® PLK1 siRNA II #12132 (+), using PLK1 (208G4) Rabbit mAb #4513 (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). The PLK1 (208G4) Rabbit mAb confirms silencing of PLK1 expression, while the β-Actin (D6A8) Rabbit mAb is used as a loading control.

Description

SignalSilence® PLK1 siRNA I from Cell Signaling Technology (CST) allows the researcher to specifically inhibit PLK1 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® PLK1 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

At least four distinct polo-like kinases exist in mammalian cells: PLK1, PLK2, PLK3, and PLK4/SAK (1). PLK1 apparently plays many roles during mitosis, particularly in regulating mitotic entry and exit. The mitosis promoting factor (MPF), cdc2/cyclin B1, is activated by dephosphorylation of cdc2 (Thr14/Tyr15) by cdc25C. PLK1 phosphorylates cdc25C at Ser198 and cyclin B1 at Ser133 causing translocation of these proteins from the cytoplasm to the nucleus (2-5). PLK1 phosphorylation of Myt1 at Ser426 and Thr495 has been proposed to inactivate Myt1, one of the kinases known to phosphorylate cdc2 at Thr14/Tyr15 (6). Polo-like kinases also phosphorylate the cohesin subunit SCC1, causing cohesin displacement from chromosome arms that allow for proper cohesin localization to centromeres (7). Mitotic exit requires activation of the anaphase promoting complex (APC) (8), a ubiquitin ligase responsible for removal of cohesin at centromeres, and degradation of securin, cyclin A, cyclin B1, Aurora A, and cdc20 (9). PLK1 phosphorylation of the APC subunits Apc1, cdc16, and cdc27 has been demonstrated in vitro and has been proposed as a mechanism by which mitotic exit is regulated (10,11).Substitution of Thr210 with Asp has been reported to elevate PLK1 kinase activity and delay/arrest cells in mitosis, while a Ser137Asp substitution leads to S-phase arrest (12). In addition, while DNA damage has been found to inhibit PLK1 kinase activity, the Thr210Asp mutant is resistant to this inhibition (13). PLK1 has been reported to be phosphorylated in vivo at Ser137 and Thr210 in mitosis; DNA damage prevents phosphorylation at these sites (14).

  1. Nigg, E.A. (1998) Curr. Opin. Cell Biol. 10, 776-783.
  2. Toyoshima-Morimoto, F. et al. (2002) EMBO Rep. 3, 341-348.
  3. Toyoshima-Morimoto, F. et al. (2001) Nature 410, 215-220.
  4. Peter, M. et al. (2002) EMBO Rep. 3, 551-556.
  5. Jackman, M. et al. (2003) Nat. Cell Biol. 5, 143-148.
  6. Nakajima, H. et al. (2003) J. Biol. Chem. 278, 25277-25280.
  7. Sumara, I. et al. (2002) Mol. Cell 9, 515-525.
  8. Hauf, S. et al. (2001) Science 293, 1320-1323.
  9. Peters, J.M. (1999) Exp. Cell Res. 248, 339-349.
  10. Kraft, C. et al. (2003) EMBO J. 22, 6598-6609.
  11. Kotani, S. et al. (1998) Mol. Cell 1, 371-380.
  12. Jang, Y.J. et al. (2002) J Biol Chem 277, 44115-20.
  13. Smits, V.A. et al. (2000) Nat Cell Biol 2, 672-6.
  14. Tsvetkov, L. and Stern, D.F. (2005) Cell Cycle 4, 166-71.

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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