Anti-APC3 (Anaphase-promoting Complex Subunit 3)

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/FZR1, 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)., Anaphase-promoting complex subunit 3 (APC3) is the human homolog of Saccharomyces cerevisiae CDC27 (9) and, like APC8/CDC23 and APC6/CDC16, is a component of the tetratricopeptide (TPR) subcomplex of the APC/C. It has been demonstrated that the binding of Cdh1/FZR1 to the APC/C depends upon the presence of APC3, implying that APC/C is activated by the association of Cdh1/FZR1 with APC3, which enables APC/C to recognize the D-box of substrates (10,11). APC3 has been shown to be localized to the centrosome at all stages of the mammalian cell cycle, and to the mitotic spindle, suggesting that APC3 plays a critical role for the transition from metaphase to anaphase during mitosis (12). During mitosis, APC3 becomes phosphorylated at numerous sites. This is predicted to change the surface charge distribution significantly such that these modifications could either induce structural changes within the APC/C by altering subunit-subunit interactions or they could change the affinity for molecules that only transiently associate with the APC/C, such as Cdh1/FZR1 (13,14). Applications: Suitable for use in Western Blot and Immunoprecipitation. Other applications not tested. Recommended Dilution: Western Blot: 1:1000. Incubate membrane with diluted antibody in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween-20 at 4°C with gentle shaking, overnight., Immunoprecipitation: 1:100, Optimal dilutions to be determined by the researcher. Storage and Stability: May be stored at 4°C for short-term only. Aliquot to avoid repeated freezing and thawing. Store at -20°C. Aliquots are stable for 12 months. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.