Cell Signaling Technology

Product Pathways - Cell Cycle / Checkpoint

Phospho-p57 Kip2 (Thr310) Antibody #2558

Applications Reactivity Sensitivity MW (kDa) Source
W IF-IC H Endogenous 57 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-p57 Kip2 (Thr310) Antibody detects endogenous levels of p57 Kip2 only when phosphorylated at threonine 310. This antibody may cross-react with p27 Kip1 when phosphorylated at Thr187.

Source / Purification

Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr310 of human p57 Kip2. Antibodies are purified by protein A and peptide affinity chromatography.

Western Blotting

Western Blotting

Western blot analysis of extracts from untreated and dexamethasone-treated (50 nM, 16 h) HeLa cells, using Phospho-p57 Kip2 (Thr310) Antibody (upper) and p57 Kip2 Antibody #2557 (lower). Lysates were treated with lambda phosphatase NEB#P0753 (10,000 U/ml for 30 mins) to confirm the phospho-specificity of the phospho-antibody.

IF-IC

IF-IC

Confocal immunofluorescent analysis of HeLa cells, dexamethasone-treated (left) or phosphatase-treated (right), labeled with Phospho-p57 Kip2 (Thr310) Antibody (green). Actin filaments have been labeled with Alexa Fluor® 555 phalloidin (red).

Background

p27 Kip1 is a member of the Cip/Kip family of cyclin-dependent kinase inhibitors. Like its relatives, p57 Kip2 and p21 Waf1/Cip1, the ability to enforce the G1 restriction point is derived from its inhibitory binding to CDK2/cyclin E and other CDK/cyclin complexes. Expression levels of p27 are upregulated in quiescent cells and in cells treated with cAMP or other negative cell cycle regulators. Downregulation of p27 can be induced by treatment with interleukin-2 or other mitogens; this involves phosphorylation of p27 and its degradation by the ubiquitin-proteasome pathway (1-4).

Levels of p57 Kip2 are controlled by ubiquitination/degradation via the Skp1/Cul1/F-box-type E3 ubiquitin ligase complex SCF-Skp2, and this effect is dependent on Thr310 (5). A similar threonine phosphorylation site in p27 Kip1, Thr187, has also been shown to regulate protein stability (6).

  1. Lloyd, R.V. et al. (1999) Am. J. Pathol. 154, 313-323.
  2. Polyak, K. et al. (1994) Genes Dev. 8, 9-22.
  3. Kato, J.Y. et al. (1994) Cell 79, 487-496.
  4. Vlach, J. et al. (1997) EMBO J. 16, 5334-5344.
  5. Kamura, T. et al. (2003) Proc. Natl. Acad. Sci. USA 100, 10231-10236.
  6. Ishida, N. et al. (2000) J. Biol. Chem. 275, 25146-25154.

Application References

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Companion Products

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

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