Revision 2

#12738Store at -20C

 

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Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Product Includes Product # Quantity Mol. Wt Isotype/Source
Phospho-CENP-A (Ser7) Antibody 2187 20 µl 17 kDa Rabbit 
Phospho-Histone H3 (Ser10) (D2C8) XP® Rabbit mAb 3377 20 µl 17 kDa Rabbit IgG
Phospho-Histone H3 (Ser28) Antibody 9713 20 µl 17 kDa Rabbit 
Phospho-p53 (Ser315) Antibody 2528 20 µl 53 kDa Rabbit 
Phospho-PLK1 (Thr210) (D5H7) Rabbit mAb 9062 20 µl 62 kDa Rabbit IgG
Phospho-TACC3 (Ser558) (D8H10) XP® Rabbit mAb 8842 20 µl 140 kDa Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl Goat 

Please visit cellsignal.com for individual component applications, species cross-reactivity, dilutions, protocols, and additional product information.

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 two western blots per primary antibody.

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

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).
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).

  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.

Background References

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    Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
    仅供研究使用。不得用于诊断流程。
    Revision 2
    #12738

    Aurora A/B Substrate Antibody Sampler Kit

    Aurora A/B Substrate Antibody Sampler Kit: Image 1 Expand Image
    使用 Phospho-CENP-A (Ser7) Antibody（上图）或 CENP-A Antibody #2186（下图），对停留在 S 期或有丝分裂的 HeLa 细胞提取物进行蛋白质印迹分析。处于 S 期的细胞用胸腺嘧啶核苷 (2 mM) 处理 12 小时，并洗涤 3 次，放入正常生长培养基 10 小时，额外用胸腺嘧啶核苷处理 12 小时，然后收集细胞。有丝分裂的细胞用胸腺嘧啶核苷处理 12 小时，并洗涤 3 次，再用 paxitaxel（终浓度 500 nM）处理 16 小时。
    Aurora A/B Substrate Antibody Sampler Kit: Image 2 Expand Image
    使用 Phospho-p53 (Ser315) Antibody（上）或 p53 Antibody #9282（下），对 MCF-7 细胞提取物进行蛋白质印迹分析，而 MCF-7 细胞为未处理或使用 nocodazole ( 50 ng/ml 处理 48 小时) 处理，或使用 nocodazole 加上 Lambda Phosphatase NEB#P0753（10,000 Units/ml, 1 小时）进行处理。
    Aurora A/B Substrate Antibody Sampler Kit: Image 3 Expand Image
    使用 Phospho-Histone H3 (Ser10) (D2C8) XP® Rabbit mAb #3377（上）或 Histone H3 Antibody #9715（下），对未处理或使用 Nocodazole（100 ng/ml 处理 18 小时）处理的 HeLa 细胞提取物，进行蛋白质印迹分析。该抗体的磷酸特异性的显示是进一步使用 λ-磷酸酶处理细胞裂解物。
    Aurora A/B Substrate Antibody Sampler Kit: Image 4 Expand Image
    一抗与靶标蛋白结合之后，与偶联 HRP 的二抗形成复合体。添加 LumiGLO®，在酶催化分解期间发光。
    Aurora A/B Substrate Antibody Sampler Kit: Image 5 Expand Image
    使用 Phospho-TACC3 (Ser558) (D8H10) XP® Rabbit mAb 对未处理的 (-) 或用胸腺嘧啶阻断同步在有丝分裂（2 mM，17 小时）后用 nocodazole 处理（100 ng/ml，24 小时）(+) 的 HT-29 细胞的提取物进行蛋白印迹分析。
    Aurora A/B Substrate Antibody Sampler Kit: Image 6 Expand Image
    使用 Phospho-PLK1 (Thr210) (D5H7) Rabbit mAb（上）或总的 PLK1 (208G4) Rabbit mAb #4513（下），对在有丝分裂中未同步或同步的 HeLa 细胞提取物进行蛋白质印迹分析。有丝分裂同步是通过胸腺嘧啶阻断进行的，随后释放进入 Nocodazole 和有丝分裂摆脱。
    Aurora A/B Substrate Antibody Sampler Kit: Image 7 Expand Image
    对未处理或使用 100 ng/ml nocodazole 处理 4 小时后在分裂中期同步的 CHO 和 HeLa 细胞提取物，进行蛋白质印迹分析，随后通过有丝分裂的摆脱作用分离中期细胞。蛋白印迹用 Phospho-Histone H3 (Ser28) Antibody #9713（上）或 Histone H3 Antibody #9715（下）探测。
    Aurora A/B Substrate Antibody Sampler Kit: Image 8 Expand Image
    使用 Phospho-CENP-A (Ser7) Antibody（绿色荧光，在合成图像中似乎呈白色）和 β-Tubulin (9F3) Rabbit mAb (Alexa Fluor® 555 Conjugate) #2116（红色）对有丝分裂的 HeLa 细胞进行共聚焦免疫荧光分析。Phospho-CENP-A 信号定位于中期平板中的亮斑。蓝色伪彩 = DRAQ5® #4084（DNA 荧光染料）。
    Aurora A/B Substrate Antibody Sampler Kit: Image 9 Expand Image
    使用 Phospho-Histone H3 (Ser10) (D2C8) XP® Rabbit mAb（绿色），对 HeLa 细胞进行共聚焦免疫荧光分析。肌动蛋白纤丝用 DY-554 Phalloidin 进行标记（红色）。蓝色伪彩 = DRAQ5® #4084（DNA 荧光染料）。
    Aurora A/B Substrate Antibody Sampler Kit: Image 10 Expand Image
    使用 Phospho-TACC3 (Ser558) (D8H10) XP® Rabbit mAb（绿色），对未处理（左）或使用 λ 磷酸酶处理（右）的 HT-29 细胞进行共聚焦免疫荧光分析。蓝色伪彩 = DRAQ5® #4084（DNA 荧光染料）。
    Aurora A/B Substrate Antibody Sampler Kit: Image 11 Expand Image
    使用 Phospho-PLK1 (Thr210) (D5H7) Rabbit mAb，对在有丝分裂中未同步或同步的 HeLa 细胞提取物进行蛋白质印迹分析。该抗体按说明用 PLK1 phospho-Thr210 肽或非磷酸化肽进行预孵育。有丝分裂同步是通过胸腺嘧啶阻断进行的，随后释放进入 Nocodazole 和有丝分裂摆脱。
    Aurora A/B Substrate Antibody Sampler Kit: Image 12 Expand Image
    使用 Phospho-Histone H3 (Ser28) Antibody（绿色），对出生后 1 天的大鼠脑进行共聚焦免疫荧光分析。蓝色伪彩 = DRAQ5™ （DNA 荧光染料）。
    Aurora A/B Substrate Antibody Sampler Kit: Image 13 Expand Image
    与碘化丙啶（DNA 含量）相比较，使用 Phospho-Histone H3 (Ser10) (D2C8) XP® Rabbit mAb，对 Jurkat 细胞进行流式细胞分析。框内细胞群是 Phospho-Histone H3 (Ser10) 阳性细胞。
    Aurora A/B Substrate Antibody Sampler Kit: Image 14 Expand Image
    使用 Phospho-Histone H3 (Ser28) Antibody（绿色），对 C2C12 细胞进行共聚焦免疫荧光分析。肌动蛋白纤丝用 Alexa Fluor®555 phalloidin（红色）进行标记。蓝色伪彩 = DRAQ5™ （DNA 荧光染料）。
    Aurora A/B Substrate Antibody Sampler Kit: Image 15 Expand Image
    与碘化丙啶（DNA 含量）相比较，使用 Phospho-Histone H3 (Ser28) Antibody 对未处理的 Jurkat 细胞进行流式细胞分析。框内细胞群是 phospho-Histone H3 阳性细胞。
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    Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
    仅供研究使用。不得用于诊断流程。