SignalSilence®APC1 siRNA II from Cell Signaling Technology (CST) allows the researcher to specifically inhibit APC1 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®APC1 siRNA II 48 to 72 hours prior to cell lysis. For transfection procedure, follow the 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 carboxy-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).
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