Cell Signaling Technology

Product Pathways - Cell Cycle / Checkpoint

Phospho-cdc25C (Thr48) Antibody #9527

Applications Reactivity Sensitivity MW (kDa) Source
W IF-IC H Endogenous 80 Rabbit

Applications Key:  W=Western Blotting  IF-IC=Immunofluorescence (Immunocytochemistry)
Reactivity Key:  H=Human
Species cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Protocols

Specificity / Sensitivity

Phospho-cdc25C (Thr48) Antibody detects endogenous levels of cdc25C only when phosphorylated at Thr48.

Source / Purification

Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr48 of human cdc25C. Antibodies are purified by protein A and peptide affinity chromatography.

Western Blotting

Western Blotting

Western blot analysis of extracts from HT29 cells, untreated (cont.) and nocodazole-treated (M), and of GST-cdc25C fusion protein, nonphosphorylated (-P) or phosphorylated (+P) by cdc2/cyclin B (New England Biolabs #P6020), using Phospho-cdc25C (Thr48) Antibody.

Western Blotting

Western Blotting

Western Blot analysis of HT29 cell extracts untreated, nocodazole-treated and lambda phosphotase-treated using Phospho-cdc25C (Thr48) (upper), and cdc25C (5H9) Rabbit mAb, #4688 (lower).

IF-IC

IF-IC

Confocal immunofluorescent analysis of HT-29 cells, untreated (left) or λ phosphatase-treated (right), using Phospho-cdc25C (Thr48) Antibody (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).


Background

cdc25 is a protein phosphatase responsible for dephosphorylating and activating cdc2, a crucial step in regulating the entry of all eukaryotic cells into mitosis (1). cdc25C is constitutively phosphorylated at Ser216 throughout interphase by c-TAK1, while phosphorylation at this site is DNA damage-dependent at the G2/M checkpoint (2). When phosphorylated at Ser216, cdc25C binds to members of the 14-3-3 family of proteins, sequestering cdc25C in the cytoplasm preventing premature mitosis (3). The checkpoint kinases Chk1 and Chk2 phosphorylate cdc25C at Ser216 in response to DNA damage (4,5).

Full activation of cdc25C involves phosphorylation at more than 12 different sites by cdc2/cyclin B and Polo-like kinase, and the activity of Pin1, a peptidyl-prolyl isomerase (PPI) (6,7). Pin1 contains a WW domain that binds phospho-Ser/Thr-Pro sites and a catalytic PPI region that induces a cis/trans isomerization on phospho-Ser/Thr-Pro bonds (8). Thr48 and Thr67 of cdc25C interact directly with the WW domain of Pin1 when these sites are phosphorylated (9). Thr48 phosphorylation also mediates binding to CKS/p13SUC1 (10).

  1. Jessus, C. and Ozon, R. (1995) Prog. Cell Cycle Res. 1, 215-228.
  2. Peng, C.Y. et al. (1997) Science 277, 1501-1505.
  3. Kumagai, A. and Dunphy, W.G. (1999) Genes Dev. 13, 1067-1072.
  4. Blasina, A. et al. (1999) Curr. Biol. 9, 1-10.
  5. Furnari, B. et al. (1999) Mol. Biol. Cell 10, 833-845.
  6. Izumi, T. and Maller, J.L. (1993) Mol. Biol. Cell 4, 1337-1350.
  7. Stukenberg, P. T. et al. (2001) Mol. Cell 7, 1071-1083.
  8. Yaffe, M. B. et al. (1997) Science 278, 1957-1960.
  9. Lu, P. J. et al. (1999) Science 283, 1325-1328.
  10. Landrieu, I. et al. (2001) J. Biol. Chem 276, 1434-1438.

Application References

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This product is intended for research purposes only. The product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

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