Revision 1

#96882Store at -20C

1 Kit

(6 x 20 microliters)

Cell Signaling Technology

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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-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
Phospho-Aurora A (Thr288)/Aurora B (Thr232)/Aurora C (Thr198) (D13A11) XP® Rabbit mAb 2914 20 µl 35, 40, 48 kDa Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl Goat 

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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 includes enough antibodies to perform two western blot experiments with each 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 antibodies.

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).
Aurora A phosphorylation of Thr210 on PLK promotes mitotic entry following checkpoint-dependent cell cycle arrest (13). Autophosphorylation of conserved threonine residues Thr288 (Aurora A), Thr232 (Aurora B), and Thr195 (Aurora C) is required for Aurora kinase activity (14).

  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. Macůrek, L. et al. (2008) Nature 455, 119-23.
  14. Willems, E. et al. (2018) Cell Div 13, 7.

Background References

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    Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
    For Research Use Only. Not for Use in Diagnostic Procedures.
    Revision 1
    #96882

    Aurora A/B Substrate Antibody Sampler Kit

    Aurora A/B Substrate Antibody Sampler Kit: Image 1 Expand Image
    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 paxitaxel (500 nM final).
    Aurora A/B Substrate Antibody Sampler Kit: Image 2 Expand Image
    Western blot analysis of extracts from HeLa, L929 and C6 cells, treated with 4 mM hydroxyurea for 20 hours to induce G1/S phase or treated with 100 nM paclitaxel or 100 ng/ml nocodazole for 20 hours to induce G2/M phase, using Phospho-Aurora A (Thr288)/Aurora B (Thr232)/Aurora C (Thr198) (D11A13) XP® Rabbit mAb (upper) or Aurora B/AIM1 Antibody #3094 (lower).
    Aurora A/B Substrate Antibody Sampler Kit: Image 3 Expand Image
    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.
    Aurora A/B Substrate Antibody Sampler Kit: Image 4 Expand Image
    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.
    Aurora A/B Substrate Antibody Sampler Kit: Image 5 Expand Image
    Western blot analysis of extracts from HT-29 cells, untreated (-) or synchronized in mitosis by thymidine block (2 mM, 17 hr) followed by nocodazole treatment (100 ng/ml, 24 hr) (+), using Phospho-TACC3 (Ser558) (D8H10) XP® Rabbit mAb.
    Aurora A/B Substrate Antibody Sampler Kit: Image 6 Expand Image
    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.
    Aurora A/B Substrate Antibody Sampler Kit: Image 7 Expand Image
    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).
    Aurora A/B Substrate Antibody Sampler Kit: Image 8 Expand Image
    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).
    Aurora A/B Substrate Antibody Sampler Kit: Image 9 Expand Image
    Confocal immunofluorescent analysis of HT-1080 cells using Phospho-Aurora A (Thr288)/Aurora B (Thr232)/Aurora C (Thr198) (D13A11) XP® Rabbit mAb (green), β-Tubulin (9F3) Rabbit mAb (Alexa Fluor® 555 Conjugate) #2116 (red), and Phospho-Histone H3 (Ser10) (6G3) Mouse mAb #9706 (blue).
    Aurora A/B Substrate Antibody Sampler Kit: Image 10 Expand Image
    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).
    Aurora A/B Substrate Antibody Sampler Kit: Image 11 Expand Image
    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).
    Aurora A/B Substrate Antibody Sampler Kit: Image 12 Expand Image
    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.
    Aurora A/B Substrate Antibody Sampler Kit: Image 13 Expand Image
    Confocal immunofluorescent analysis of postnatal day 1 rat brain using Phospho-Histone H3 (Ser28) Antibody (green). Blue pseudocolor = DRAQ5™ (fluorescent DNA dye).
    Aurora A/B Substrate Antibody Sampler Kit: Image 14 Expand Image
    Flow cytometric analysis of untreated Jurkat cells using Phospho-Aurora A (Thr288)/Aurora B (Thr232)/Aurora C (Thr198) (D13A11) XP® Rabbit mAb compared to propidium iodide (DNA content). The boxed population indicates phospho-Aurora A (Thr288)/Aurora B (Thr232)/Aurora C (Thr198)-positive cells.
    Aurora A/B Substrate Antibody Sampler Kit: Image 15 Expand Image
    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.
    Aurora A/B Substrate Antibody Sampler Kit: Image 16 Expand Image
    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).
    Aurora A/B Substrate Antibody Sampler Kit: Image 17 Expand Image
    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.
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    Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
    For Research Use Only. Not for Use in Diagnostic Procedures.