#12738 Aurora A/B Substrate Antibody Sampler Kit
|Phospho-CENP-A (Ser7) Antibody #2187||20 µl||WB, IP, IF-IC||H||Mk||17||Rabbit|
|Phospho-Histone H3 (Ser10) (D2C8) XP® Rabbit mAb #3377||20 µl||WB, IF-IC, F||H, M, R, Mk, Z||17||Rabbit IgG|
|Phospho-Histone H3 (Ser28) Antibody #9713||20 µl||WB, IP, IF-F, IF-IC, F||H, M, Hm, Dm||R, C, X, Z, B||17||Rabbit|
|Phospho-p53 (Ser315) Antibody #2528||20 µl||WB||H||Mk, B||53||Rabbit|
|Phospho-PLK1 (Thr210) (D5H7) Rabbit mAb #9062||20 µl||WB, IP||H||62||Rabbit IgG|
|Phospho-TACC3 (Ser558) (D8H10) XP® Rabbit mAb #8842||20 µl||WB, IF-IC||H||140||Rabbit IgG|
|Anti-rabbit IgG, HRP-linked Antibody #7074||100 µl||WB||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
After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.
Western blot analysis of extracts from MCF-7 cells, untreated or treated with nocodazole (50 ng/ml, 48h) or nocodazole plus Lambda Phosphatase NEB#P0753 (10,000 Units/ml, 1h), using Phospho-p53 (Ser315) Antibody (upper) or p53 Antibody #9282 (lower).
Western blot analysis of lysates from CHO and HeLa cells either untreated or synchronized in metaphase by treatment with 100 ng/ml nocodazole for 4 h, followed by isolation of metaphase cells by mitotic shake-off. Blots were probed with Phospho-Histone H3 (Ser28) Antibody #9713 (upper) or Histone H3 Antibody #9715 (lower).
Western blot analysis of extracts from HeLa cells arrested in S phase or mitosis using Phospho-CENP-A (Ser7) Antibody (upper panel) or CENP-A Antibody #2186 (lower panel). S phase cells were treated for 12 hours with thymidine (2 mM), 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 HeLa cells, either untreated or treated with nocodazole (100 ng/ml for 18 hours), using Phospho-Histone H3 (Ser10) (D2C8) XP® Rabbit mAb #3377 (upper) or Histone H3 Antibody #9715 (lower). Phospho-specificity of the antibody is shown by further treatment of the lysate with λ phosphatase.
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.
Western blot analysis of extracts from HeLa cells, asynchronous or synchronized in mitosis, using Phospho-PLK1 (Thr210) (D5H7) Rabbit mAb (upper) or total PLK1 (208G4) Rabbit mAb #4513 (lower). Mitotic synchronization was performed by thymidine block followed by release into nocodazole and mitotic shake-off.
Flow cytometric analysis of untreated Jurkat cells, using Phospho-Histone H3 (Ser28) Antibody versus propidium iodide (DNA content). The box indicates phospho-Histone H3 positive cells.
Flow cytometric analysis of Jurkat cells using Phospho-Histone H3 (Ser10) (D2C8) XP® Rabbit mAb versus propidium iodide (DNA content). The boxed population indicates Phospho-Histone H3 (Ser10) positive cells.
Confocal immunofluorescent analysis of HT-29 cells, untreated (left) or λ phosphatase-treated (right), using Phospho-TACC3 (Ser558) (D8H10) XP® Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Western blot analysis of extracts from HeLa cells, asynchronous or synchronized in mitosis, using Phospho-PLK1 (Thr210) (D5H7) Rabbit mAb. The antibody was pre-incubated with a PLK1 phospho-Thr210 peptide or nonphospho-peptide as indicated. Mitotic synchronization was performed by thymidine block followed by release into nocodazole and mitotic shake-off.
Confocal immunofluorescent analysis of a mitotic HeLa cell using Phospho-CENP-A (Ser7) Antibody (green fluorescence, appearing as white in the composite image) and β-Tubulin (9F3) Rabbit mAb (Alexa Fluor® 555 Conjugate) #2116 (red). Phospho-CENP-A signal is localized to bright spots in the metaphase plate. Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Confocal immunofluorescent analysis of C2C12 cells using Phospho-Histone H3 (Ser28) Antibody (green). Actin filaments have been labeled with Alexa Fluor® 555 phalloidin (red). Blue pseudocolor = DRAQ5™ (fluorescent DNA dye).
Confocal immunofluorescent analysis of HeLa cells using Phospho-Histone H3 (Ser10) (D2C8) XP® Rabbit mAb (green). Actin filaments have been labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Confocal immunofluorescent analysis of postnatal day 1 rat brain using Phospho-Histone H3 (Ser28) Antibody (green). Blue pseudocolor = DRAQ5™ (fluorescent DNA dye).
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).
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DRAQ5 is a registered trademark of Biostatus Limited.
XP is a registered trademark of Cell Signaling Technology, Inc.
Alexa Fluor is a registered trademark of Life Technologies Corporation.
LumiGLO is a registered trademark of Kirkegaard & Perry Laboratories.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.