Cell Signaling Technology
XP Monoclonal Antibody

Product Pathways - Cell Cycle / Checkpoint

Phospho-Cyclin D1 (Thr286) (D29B3) XP® Rabbit mAb #3300

Applications Reactivity Sensitivity MW (kDa) Isotype
W IP IF-IC F H (Mk) Endogenous 36 Rabbit IgG

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IF-IC=Immunofluorescence (Immunocytochemistry)  F=Flow Cytometry
Reactivity Key:  H=Human  Mk=Monkey
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-Cyclin D1 (Thr286) (D29B3) XP® Rabbit mAb detects endogenous levels of cyclin D1 only when phosphorylated at Thr286. The antibody does not cross-react with other cyclin D family members.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr286 of cyclin D1.

Western Blotting

Western Blotting

Western blot analysis of extracts from HT-1080 cells, treated with MG132 and with or without λ phosphatase, using Phospho-Cyclin D1 (Thr286) (D29B3) XP® Rabbit mAb (upper) or Cyclin D1 (DC56) Mouse mAb #2926 (lower).

Western Blotting

Western Blotting

Western blot analysis of extracts from HT-1080 cells, untreated or treated with MG132 (10 μM, 4 hours), using Phospho-Cyclin D1 (Thr286) (D29B3) XP® Rabbit mAb.

Flow Cytometry

Flow Cytometry

Flow cytometric analysis of HT-1080 cells, untreated (blue) or MG132-treated (green), using Phospho-Cyclin D1 (Thr286) (D29B3) XP® Rabbit mAb.


IF-IC

IF-IC

Confocal immmunofluorescent analysis of HT-1080 (top) and Saos-2 cells (bottom), untreated (left) or treated with MG132 alone (center) or with MG132 followed by λ-phosphatase (right), using Phospho-Cyclin D1 (Thr286) (D29B3) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red).

Background

Activity of the cyclin-dependent kinases CDK4 and CDK6 is regulated by T-loop phosphorylation, by the abundance of their cyclin partners (the D-type cyclins), and by association with CDK inhibitors of the Cip/Kip or INK family of proteins (1). The inactive ternary complex of cyclin D/CDK4 and p27 Kip1 requires extracellular mitogenic stimuli for the release and degradation of p27 concomitant with a rise in cyclin D levels to affect progression through the restriction point and Rb-dependent entry into S-phase (2). The active complex of cyclin D/CDK4 targets the retinoblastoma protein for phosphorylation, allowing the release of E2F transcription factors that activate G1/S-phase gene expression (3). Levels of cyclin D protein drop upon withdrawal of growth factors through downregulation of protein expression and phosphorylation-dependent degradation (4).

Aberrant expression of cyclin D1 is associated with many forms of cancer, including B cell lymphomas. Gene translocation or amplification of the cyclin D1 gene can directly contribute to oncogenesis (2). Cyclin D1 also plays a critical role in mammary tissue maturation (5). Phosphorylation of cyclin D1 at Thr286 by glycogen synthase kinase 3β (4) or through the Ras/Raf/MEK/MAPK pathway (6) enhances its ubiquitination and proteasomal degradation.

  1. Hirai, H. et al. (1995) Mol. Cell. Biol. 15, 2672-2681.
  2. Sherr, C.J. (1996) Science 274, 1672-1677.
  3. Lukas, J. et al. (1996) Mol. Cell. Biol. 16, 6917-6925.
  4. Diehl, J.A. et al. (1997) Genes Dev. 11, 957-972.
  5. Sicinski, P. et al. (1995) Cell 82, 621-630.
  6. Shao, J. et al. (2000) J Biol Chem 275, 22916-24.

Application References

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

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