Phospho-PLK1 (Thr210) (D5H7) R

9062:

Immunoprecipitation , Western Blotting

Phospho-PLK1 (Thr210) (D5H7) Rabbit mAb recognizes endogenous levels of PLK1 protein only when phosphorylated at Thr210.

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Thr210 of human PLK1 protein.

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 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.

At least 4 distinct polo-like kinases exist in mammalian cells: PLK1, PLK2, PLK3, and PLK4/SAK (1). PLK1 apparently plays many roles during mitosis, particularly in regulating mitotic entry and exit. The mitosis promoting factor (MPF), cdc2/cyclin B1, is activated by dephosphorylation of cdc2 (Thr14/Tyr15) by cdc25C. PLK1 phosphorylates cdc25C at Ser198 and cyclin B1 at Ser133 causing translocation of these proteins from the cytoplasm to the nucleus (2-5). PLK1 phosphorylation of Myt1 at Ser426 and Thr495 has been proposed to inactivate Myt1, one of the kinases known to phosphorylate cdc2 at Thr14/Tyr15 (6). Polo-like kinases also phosphorylate the cohesin subunit SCC1, causing cohesin displacement from chromosome arms that allow for proper cohesin localization to centromeres (7). Mitotic exit requires activation of the anaphase promoting complex (APC) (8), a ubiquitin ligase responsible for removal of cohesin at centromeres, and degradation of securin, cyclin A, cyclin B1, Aurora A, and cdc20 (9). PLK1 phosphorylation of the APC subunits Apc1, cdc16, and cdc27 has been demonstrated in vitro and has been proposed as a mechanism by which mitotic exit is regulated (10,11).Substitution of Thr210 with Asp has been reported to elevate PLK1 kinase activity and delay/arrest cells in mitosis, while a Ser137Asp substitution leads to S-phase arrest (12). In addition, while DNA damage has been found to inhibit PLK1 kinase activity, the Thr210Asp mutant is resistant to this inhibition (13). PLK1 has been reported to be phosphorylated in vivo at Ser137 and Thr210 in mitosis; DNA damage prevents phosphorylation at these sites (14).

1. Nigg, E.A. (1998) Curr. Opin. Cell Biol. 10, 776-783.
2. Toyoshima-Morimoto, F. et al. (2002) EMBO Rep. 3, 341-348.
3. Toyoshima-Morimoto, F. et al. (2001) Nature 410, 215-220.
4. Peter, M. et al. (2002) EMBO Rep. 3, 551-556.
5. Jackman, M. et al. (2003) Nat. Cell Biol. 5, 143-148.
6. Nakajima, H. et al. (2003) J. Biol. Chem. 278, 25277-25280.
7. Sumara, I. et al. (2002) Mol. Cell 9, 515-525.
8. Hauf, S. et al. (2001) Science 293, 1320-1323.
9. Peters, J.M. (1999) Exp. Cell Res. 248, 339-349.
10. Kraft, C. et al. (2003) EMBO J. 22, 6598-6609.
11. Kotani, S. et al. (1998) Mol. Cell 1, 371-380.
12. Jang, Y.J. et al. (2002) J Biol Chem 277, 44115-20.
13. Smits, V.A. et al. (2000) Nat Cell Biol 2, 672-6.
14. Tsvetkov, L. and Stern, D.F. (2005) Cell Cycle 4, 166-71.

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• 4513 PLK1 (208G4) Rabbit mAb
• 7071 Phototope^® -HRP Western Blot Detection System, Anti-rabbit IgG, HRP-linked Antibody
• 7074 Anti-rabbit IgG, HRP-linked Antibody
• 7720 Prestained Protein Marker, Broad Range (Premixed Format)
• 7727 Biotinylated Protein Ladder Detection Pack
• 7003 20X LumiGLO^® Reagent and 20X Peroxide

For Research Use Only. Not For Use In Diagnostic Procedures.