Cell Signaling Technology

Product Pathways - Cell Cycle / Checkpoint

Aurora A/B Substrate Antibody Sampler Kit #12738

REACTIVITY

No. Size Price
12738T 1 Kit ( 6 x 20 µl ) ¥5,301.00 现货查询 购买询价
Kit Includes Quantity Applications Reactivity Homology† MW (kDa) Isotype
Phospho-CENP-A (Ser7) Antibody #2187 20 µl W,IP,IF-IC, H, Mk, 17 Rabbit
Phospho-p53 (Ser315) Antibody #2528 20 µl W, H, Mk,B, 53 Rabbit
Phospho-Histone H3 (Ser10) (D2C8) XP® Rabbit mAb #3377 20 µl W,IF-IC,F, H,M,R,Mk,Z, 17 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody #7074 100 µl W, Goat
Phospho-TACC3 (Ser558) (D8H10) XP® Rabbit mAb #8842 20 µl W,IF-IC, H, 140 Rabbit IgG
Phospho-PLK1 (Thr210) (D5H7) Rabbit mAb #9062 20 µl W,IP, H, 62 Rabbit IgG
Phospho-Histone H3 (Ser28) Antibody #9713 20 µl W,IP,IF-F,IF-IC,F, H,M,Hm,Dm, R,C,X,Z,B, 17 Rabbit

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.

Aurora Antibody Sampler Kit中的每个抗体都能检测各自内源性的靶蛋白,而不与其他家族成员发生交叉反应。

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.

多抗由合成肽段免疫动物产生,这些肽段分别与人CENP-A蛋白Ser7附近氨基酸,人p53蛋白的Ser315附近氨基酸,人PLK1蛋白的Thr210附近氨基酸和Ser28磷酸化的组蛋白的羧基端附近氨基酸一一对应。抗体由蛋白A和肽段亲和层析技术纯化得到。磷酸特异性单抗由合成肽段免疫动物产生,该肽段分别与人组蛋白H3的Ser10邻近氨基酸和人TACC3蛋白的Ser558邻近氨基酸一致。

Description

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.

Aurora A/B Substrate Antibody Sampler Kit提供了一种经济的方式去检测细胞的G2/M期。该试剂盒提供了足够的一抗用于四次western blot实验。

Western Blotting

Western Blotting

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 Blotting

Western Blotting

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 Blotting

Western Blotting

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).

IF-IC

IF-IC

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).

IF-F

IF-F

Confocal immunofluorescent analysis of postnatal day 1 rat brain using Phospho-Histone H3 (Ser28) Antibody (green). Blue pseudocolor = DRAQ5™ (fluorescent DNA dye).

Flow Cytometry

Flow Cytometry

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.

Western Blotting

Western Blotting

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).

Flow Cytometry

Flow Cytometry

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.

IF-IC

IF-IC

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).

Western Blotting

Western Blotting

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.

IF-IC

IF-IC

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 Blotting

Western Blotting

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 Blotting

Western Blotting

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.

Western Blotting

Western Blotting

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.

Western Blotting

Western Blotting

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).CHO和HeLa细胞,未处理(-)或诺考达唑(100 ng/ml, 4 hr; +)处理同步在中期随后使用mitotic shake-off分离处于中期的细胞,获得它们的细胞提取物,使用Phospho-Histone H3 (Ser28) Antibody #9713(上)或Histone H3 Antibody #9715(下)进行western blot分析。

Western Blotting

Western Blotting

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).未处理(-)或使用诺考达唑(100 ng/ml, 18 hr; +)或 λ phosphatase处理的HeLa细胞提取物,使用Phospho-Histone H3 (Ser10) (D2C8) XP®Rabbit mAb #3377(上)或Histone H3 Antibody #9715(下)进行western blot分析。

Western Blotting

Western Blotting

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. 异步或同步在分裂期的HT-29细胞提取物,使用Phospho-PLK1 (Thr210) Antibody #5472进行western blot分析。抗体预先与PLK1 phospho-Thr210 peptide或nonphospho-peptide进行孵育(上)。同样的裂解液也使用total PLK1 (208G4) Rabbit mAb #4513进行检测(下)。胸苷抑制随后使用诺考达唑处理再进行mitotic shake-off能够使细胞同步于有丝分裂期。

Western Blotting

Western Blotting

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.未处理(-)或使用诺考达唑(50 ng/ml, 48 hr; +)或 λ phosphatase (10,000 units/ml, 1hr; +)处理的MCF7细胞提取物,使用Phospho-p53 (Ser315) Antibody #2528进行western blot分析。

Western Blotting

Western Blotting

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.未处理(-)或胸苷抑制(2 mM, 17 hr)后经诺考达唑处理(100 ng/ml, 24 hr; +)同步在有丝分裂期的HT-29细胞提取物,使用Phospho-TACC3 (Ser558) (D8H10) XP® Rabbit mAb #8842进行western blot分析。

Western Blotting

Western Blotting

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). 对捕获在S期或有丝分裂期的HeLa细胞提取物,使用Phospho-CENP-A (Ser7) Antibody #2187 (上) 或 CENP-A Antibody #2186 (下)进行western blot分析。S期细胞经过胸苷(2 mM, 12 hr)处理,然后冲洗3次,放入正常生长培养基生长10小时再被胸苷处理12小时。有丝分裂期细胞经过胸苷处理12小时,冲洗3次,然后使用紫杉醇(500 nM final)处理16小时。

IF-IC

IF-IC

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).

Background

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). Aurora激酶属于一组高度保守的有丝分裂丝氨酸/苏氨酸激酶家族,哺乳动物中发现了三个成员:Aurora A, B,和 C (1,2). 对有丝分裂细胞中Aurora激酶的暂时表达模式以及亚细胞定位提示它们与有丝分裂的结构相关。Aurora激酶影响功能从G2期到胞质分裂器,有可能涉及了重要的细胞周期时间例如中心体复制,染色体的双向定位和隔离,卵裂沟的定位和脱离(3)。Aurora A在中心体上被发现,沿着有丝分裂的纺锤体微管和有丝分裂增殖细胞的细胞质中分布。Aurora A蛋白水平在G1和S期很低,在G2/M期达到最高。磷酸化Aurora A羧基端的Thr288会增加其激酶活性。Aurora A涉及到中心体分离,成熟和纺锤体聚集以及稳定。Aurora B蛋白也在G2/M期表达最多;Aurora B激酶主要在有丝分裂的中期到末期转换时发挥作用。前期时Aurora B在染色体定位到纺锤体之前结合。Aurora B通过控制微管-着丝粒的结合和胞质分裂调控染色体分离。G2/M期Aurora A和Aurora B的表达与组蛋白H3的磷酸化高度一致(4,5);研究人员已经发现多种人类癌症中这些基因都是高表达的(2,4)。在前期到胞质分裂期,Aurora C都位于中心体上,它的mRNA和蛋白水平都在G2/M期达到最高。尽管典型的Aurora C蛋白的表达限定在睾丸中,研究发现也在多种癌细胞中发现了过表达的Aurora C (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).

转化酸性卷曲螺旋(TACC)蛋白的常见特征是在羧基端含有一个近200氨基酸的基序。当被Aurora A 磷酸化Ser558后,哺乳动物TACC3会定位到纺锤体的位置并提高微管稳定性(8,9)。有丝分裂前期CENP-A磷酸化Ser7依赖于Aurora-A,这对于前期将Aurora B定位到内着丝粒蛋白的位置,恰当的着丝粒/微管连接和有丝分裂时染色质排列是必须的(10)。Aurora B也能协助CENP-A磷酸化Ser7,该磷酸化随后会调控胞质分裂期Aurora B的功能(11)。Aurora B能磷酸化组蛋白H3的Ser10和Ser28,着能协助有丝分裂时染色质的凝集(12)。p53的激活会导致细胞周期停滞和DNA修复以及凋亡(13)。Aurora A磷酸化p53的Ser315依赖于细胞周期,会导致MDM2介导的泛素化/p53降解(14)。PLK磷酸化Aurora的Thr210能促进检验点依赖的细胞周期停滞后进入有丝分裂使其(15)。

  1. Warner, S.L. et al. (2003) Mol Cancer Ther 2, 589-95.
  2. Katayama, H. et al. (2003) Cancer Metastasis Rev 22, 451-64.
  3. Andrews, P.D. et al. (2003) Curr Opin Cell Biol 15, 672-83.
  4. Pascreau, G. et al. (2003) Prog Cell Cycle Res 5, 369-74.
  5. Crosio, C. et al. (2002) Mol Cell Biol 22, 874-85.
  6. Kimura, M. et al. (1999) J Biol Chem 274, 7334-40.
  7. Gergely, F. et al. (2000) Proc Natl Acad Sci U S A 97, 14352-7.
  8. Kinoshita, K. et al. (2005) J Cell Biol 170, 1047-55.
  9. Schneider, L. et al. (2007) J Biol Chem 282, 29273-83.
  10. Kunitoku, N. et al. (2003) Dev Cell 5, 853-64.
  11. Zeitlin, S.G. et al. (2001) J Cell Biol 155, 1147-57.
  12. Goto, H. et al. (2002) Genes Cells 7, 11-7.
  13. Levine, A.J. (1997) Cell 88, 323-31.
  14. Sakaguchi, K. et al. (1998) Genes Dev 12, 2831-41.
  15. Macůrek, L. et al. (2008) Nature 455, 119-23.

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