Western blot analysis of extracts from 293T cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-) or SignalSilence® APC2 siRNA I (+), using APC2 Antibody #12301 (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). The APC2 Antibody confirms silencing of APC2 expression, while the β-Actin (D6A8) Rabbit mAb is used as a loading control.
CST recommends transfection with 100 nM SignalSilence® APC2 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® APC2 siRNA I from Cell Signaling Technology (CST) allows the researcher to specifically inhibit APC2 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 2 (APC2) is a distant member of the cullin family (9,10) that interacts with a RING-H2 finger protein related to Rbx1/Hrt1/Roc1, called APC11, to form the catalytic subcomplex of the APC/C. The APC2/11 subcomplex recruits E2 enzymes such as UBE2C/UBCH10 and is required for the APC/C to catalyze substrate ubiquitination (11). Therefore, APC is a member of the expanding family of cullin-RING finger-based ubiquitin ligases. The physiologic importance of APC2 was underscored by the finding that disruption of murine Apc2 causes embryonic lethality (12).
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