Cell Signaling Technology

Product Pathways - Cell Cycle / Checkpoint

SignalSilence® APC6 siRNA I #7279

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 293T cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-), SignalSilence® APC6 siRNA I (+) or SignalSilence® APC6 siRNA II #7164 (+), using APC6 (D8D8) Rabbit mAb #9499 (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). The APC6 (D8D8) Rabbit mAb confirms silencing of APC6 expression, while the GAPDH (D16H11) XP® Rabbit mAb is used as a loading control.

Description

SignalSilence® APC6 siRNA I from Cell Signaling Technology (CST) allows the researcher to specifically inhibit APC6 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® APC6 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

Cell proliferation in all eukaryotic cells depends strictly upon the ubiquitin ligase (E3) activity of the anaphase promoting complex/cyclosome (APC/C), whose main function is to trigger the transition of the cell cycle from metaphase to anaphase. APC/C is a 1.5 MDa protein complex found in the nucleus of interphase cells. This complex diffuses throughout the cytoplasm and associates with parts of the spindle apparatus during mitosis. APC/C performs its various functions by promoting the assembly of polyubiquitin chains on substrate proteins, which targets these proteins for degradation by the 26S proteasome (1,2). In humans, twelve different APC/C subunits have been identified. Like all E3 enzymes, APC/C utilizes ubiquitin residues that have been activated by E1 enzymes and then transferred to E2 enzymes. Indeed APC/C has been shown to transiently interact with UBCH5 and UBCH10 E2 enzymes, in part, via the RING-finger domain-containing subunit, APC11 (3-5). In addition to E2 enzymes, APC/C activity is also strictly dependent upon one of several cofactors that associate with APC/C during specific phases of the cell cycle. The best studied of these are Cdc20 and Cdh1, which contain a C-terminal WD40 domain and participate in the recognition of APC/C substrates by interacting with specific recognition elements in these substrates (6), called D-Boxes (7) and KEN-boxes (8).

APC6/CDC16 is a component of the tetratricopeptide repeat (TPR) sub-complex of the APC/C, which also consists of APC8/CDC23 and APC3/CDC27. It is thought that this sub-complex plays an important role in coordinating the juxtaposition of the catalytic and substrate recognition modules relative to co-activator, regulatory proteins, and substrates (9). There is also evidence suggesting that phosphorylation of APC6 and the other TPR subunits during mitosis plays a functional role in regulating the association between TPR subunits and substrate recognition subunits such as Cdc20 (10).

  1. Qiao, X. et al. (2010) Cell Cycle 9, 3904-12.
  2. Harper, J.W. et al. (2002) Genes Dev 16, 2179-206.
  3. Carroll, C.W. and Morgan, D.O. (2002) Nat Cell Biol 4, 880-7.
  4. Gmachl, M. et al. (2000) Proc Natl Acad Sci U S A 97, 8973-8.
  5. Leverson, J.D. et al. (2000) Mol Biol Cell 11, 2315-25.
  6. Kraft, C. et al. (2005) Mol Cell 18, 543-53.
  7. Glotzer, M. et al. (1991) Nature 349, 132-8.
  8. Pfleger, C.M. and Kirschner, M.W. (2000) Genes Dev 14, 655-65.
  9. Schreiber, A. et al. (2011) Nature 470, 227-32.
  10. Kraft, C. et al. (2003) EMBO J 22, 6598-609.

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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