Western blot analysis of extracts from 293T cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-) or SignalSilence® APC3 siRNA I (+), using APC3 Antibody #9063 (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). The APC3 Antibody confirms silencing of APC3 expression, while the GAPDH (D16H11) XP® Rabbit mAb is used as a loading control.
CST recommends transfection with 100 nM SignalSilence® APC3 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.
SignalSilence® siRNA is supplied in RNAse-free water. Aliquot and store at -20ºC.
SignalSilence® APC3 siRNA I from Cell Signaling Technology (CST) allows the researcher to specifically inhibit APC3 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.
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.
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 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 interact with UBE2S and UBE2C E2 enzymes, in part, via the RING-finger domain-containing subunit, APC11 (3-5). APC/C activity is also strictly dependent upon its association with multiple cofactors. For example, the related proteins, Cdc20 and Cdh1/FZR1, 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).
Anaphase-promoting complex subunit 3 (APC3), APC8, and APC6 are components of the tetratricopeptide (TPR) APC/C subcomplex (9). The presence of APC3 is required for binding of Cdh1/FZR1 to the APC/C. This suggests that APC/C is activated by an association between Cdh1/FZR1 with APC3 that enables APC/C to recognize the D-box of substrates (6,10). APC3 localizes to the centrosome and the mitotic spindle, suggesting that APC3 plays a critical role in the transition from metaphase to anaphase (11). Phosphorylation of APC3 at multiple sites during mitosis likely leads to structural changes within the APC/C by altering subunit interactions or changing affinity for molecules that transiently associate with the APC/C, such as Cdh1/FZR1 (12,13).
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