Product Pathways - Cell Cycle / Checkpoint
Aurora A/B Substrate Antibody Sampler Kit #12738
|12738S||1 Kit (6 x 40 µl)||---||In Stock||---|
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|Kit Includes||Quantity||Applications||Reactivity||Homology†||MW (kDa)||Isotype|
|Phospho-CENP-A (Ser7) Antibody #2187||40 µl||W, IP, IF-IC||H||Mk||17||Rabbit|
|Phospho-Histone H3 (Ser10) (D2C8) XP® Rabbit mAb #3377||40 µl||W, IF-IC, F||H, M, R, Mk, Z||17||Rabbit IgG|
|Phospho-Histone H3 (Ser28) Antibody #9713||40 µl||W, IP, IF-F, IF-IC, F||H, M, Hm, Dm||R, C, X, Z, B||17||Rabbit|
|Phospho-p53 (Ser315) Antibody #2528||40 µl||W||H||Mk, B||53||Rabbit|
|Phospho-PLK1 (Thr210) Antibody #5472||40 µl||W||H||Mk||62||Rabbit|
|Phospho-TACC3 (Ser558) (D8H10) XP® Rabbit mAb #8842||40 µl||W, IF-IC||H||140||Rabbit IgG|
|Anti-rabbit IgG, HRP-linked Antibody #7074||100 µl||Goat|
†Species predicted to react based on 100% sequence homology.
Applications Key: W=Western Blotting, IP=Immunoprecipitation, IF-IC=Immunofluorescence (Immunocytochemistry), F=Flow Cytometry, IF-F=Immunofluorescence (Frozen)
Reactivity Key: H=Human, M=Mouse, R=Rat, Mk=Monkey, Z=Zebrafish, Hm=Hamster, Dm=D. melanogaster
Western blot analysis of extracts from HeLa cells arrested in S phase or mitosis using Phospho-CENP-A (Ser7) Antibody #2187 (upper panel) or CENP-A Antibody #2186 (lower panel). S phase cells were treated with thymidine (2 mM, 12 hr), rinsed three times, released into normal growth medium for 10 hours and then treated an additional 12 hours with thymidine before harvesting. Mitotic cells were treated for 12 hours with thymidine, rinsed three times and then treated for 16 hours with paxitaxol (500 nM final).
Western blot analysis of extracts from MCF7 cells, untreated (-), treated with nocodazole (50 ng/ml, 48 hr; +) or λ phosphatase (10,000 units/ml, 1hr; +), using Phospho-p53 (Ser315) Antibody #2528.
Western blot analysis of extracts from HeLa cells, untreated (-), treated with nocodazole (100 ng/ml, 18 hr; +) or λ phosphatase, using Phospho-Histone H3 (Ser10) (D2C8) XP® Rabbit mAb #3377 (upper) or Histone H3 Antibody #9715 (lower).
Western blot analysis of extracts from HT-29 cells, asynchronous or synchronized in mitosis using Phospho-PLK1 (Thr210) Antibody #5472. The antibody was pre-incubated with a PLK1 phospho-Thr210 peptide or nonphospho-peptide as indicated (upper). The same lysates were examined using total PLK1 (208G4) Rabbit mAb #4513 (lower). Mitotic synchronization was performed by thymidine block followed by release into nocodazole and mitotic shake-off.
Western blot analysis of extracts from HT-29 cells, untreated (-) or synchronized in mitosis by thymidine block (2 mM, 17 hr) followed by nocodozole treatment (100 ng/ml, 24 hr; +), using Phospho-TACC3 (Ser558) (D8H10) XP® Rabbit mAb #8842.
Western blot analysis of extracts from CHO and HeLa cells, untreated (-) or synchronized in metaphase by treatment with nocodazole (100 ng/ml, 4 hr; +), followed by isolation of metaphase cells by mitotic shake-off, using Phospho-Histone H3 (Ser28) Antibody #9713 (upper) or Histone H3 Antibody #9715 (lower).
The Aurora A/B Substrate Antibody Sampler Kit provides an economical means to investigate the G2/M phase of the cell cycle. The kit contains enough primary antibody to perform four western blots per primary antibody.
Specificity / Sensitivity
Each antibody in the Aurora Antibody Sampler Kit detects endogenous levels of its respective modification-specific target protein and does not cross-react with other family members.
Source / Purification
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser7 of human CENP-A protein, residues surrounding Ser315 of human p53 protein, residues surrounding Thr210 of human PLK1 protein, and with a synthetic peptide corresponding to the amino terminus of histone H3 phosphorylated on Ser28. Polyclonal antibodies are purified by protein A and peptide affinity chromatography. Monoclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser10 of human histone H3 protein and residues surrounding Ser558 of human TACC3 protein.
Aurora kinases belong to a highly conserved family of mitotic serine/threonine kinases with three members identified among mammals: Aurora A, B, and C (1,2). Studies on the temporal expression pattern and subcellular localization of Aurora kinases in mitotic cells suggest an association with mitotic structure. Aurora kinase functional influences span from G2 phase to cytokinesis and may be involved in key cell cycle events such as centrosome duplication, chromosome bi-orientation and segregation, cleavage furrow positioning, and ingression (3). Aurora A is detected at the centrosomes, along mitotic spindle microtubules, and in the cytoplasm of mitotically proliferating cells. Aurora A protein levels are low during G1 and S phases and peak during the G2/M phase of the cell cycle. Phosphorylation of Aurora A at Thr288 in its catalytic domain increases kinase activity. Aurora A is involved in centrosome separation, maturation, and spindle assembly and stability. Expression of Aurora B protein also peaks during the G2/M phase of the cell cycle; Aurora B kinase activity peaks at the transition from metaphase to the end of mitosis. Aurora B associates with chromosomes during prophase prior to relocalizing to the spindle at anaphase. Aurora B regulates chromosome segregation through the control of microtubule-kinetochore attachment and cytokinesis. Expression of both Aurora A and Aurora B during the G2/M phase transition is tightly coordinated with histone H3 phosphorylation (4,5); research investigators have observed overexpression of these kinases in a variety of human cancers (2,4). Aurora C localizes to the centrosome from anaphase to cytokinesis and both mRNA and protein levels peak during G2/M phase. Although typical Aurora C expression is limited to the testis, research studies report overexpression of Aurora C is detected in various cancer cell lines (6).
Transforming acid coiled-coil (TACC) proteins are a family of proteins characterized by a common coiled-coil motif of approximately 200 amino acids at the carboxy-terminal end (7). When phosphorylated at Ser558 by Aurora A, mammalian TACC3 is localized to mitotic spindles and increases microtubule stability (8,9).
Aurora A-dependent phosphorylation of CENP-A on Ser7 during prophase is required for proper targeting of Aurora B to the inner centromere in prometaphase, proper kinetochore/microtubule attachment and proper alignment of chromosomes during mitosis (10). Aurora B also targets Ser7 on CENP-A, which in turn regulates Aurora B activity during cytokinesis (11). Aurora B phosphorylates both Ser10 and Ser28 on histone H3 in concordance with mitotic chromosome condensation (12).
Activation of p53 can lead to either cell cycle arrest and DNA repair or apoptosis (13). Aurora A phosphorylates p53 at Ser315 in a cell cycle-dependent manner leading to MDM2-mediated ubiquitination/degradation of p53 (14). Aurora A phosphorylation of Thr210 on PLK promotes mitotic entry following checkpoint-dependent cell cycle arrest (15).
- Warner, S.L. et al. (2003) Mol. Cancer Ther. 2, 589-595.
- Katayama , H. et al. (2003) Cancer Metastasis Rev. 22, 451-464.
- Andrews, P.D. et al. (2003) Curr. Opin. Cell Biol. 15, 672-683.
- Pascreau, G. et al. (2003) Prog. Cell Cycle Res. 5, 369-374.
- Crosio, C. et al. (2002) Mol. Cell. Biol. 22, 874-885.
- Kimura, M. et al. (1999) J. Biol. Chem. 274, 7334-7340.
- Gergely, F. et al. (2000) Proc Natl Acad Sci U S A 97, 14352-7.
- Kinoshita, K. et al. (2005) J Cell Biol 170, 1047-55.
- Schneider, L. et al. (2007) J Biol Chem 282, 29273-83.
- Kunitoku, N. et al. (2003) Dev Cell 5, 853-64.
- Zeitlin, S.G. et al. (2001) J Cell Biol 155, 1147-57.
- Goto, H. et al. (2002) Genes Cells 7, 11-7.
- Levine, A.J. (1997) Cell 88, 323-31.
- Sakaguchi, K. et al. (1998) Genes Dev 12, 2831-41.
- Macůrek, L. et al. (2008) Nature 455, 119-23.
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* Product-specific protocol.
For Research Use Only. Not For Use In Diagnostic Procedures.
XP® is a trademark of Cell Signaling Technology, Inc.
Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc.