Cell Signaling Technology
XP Monoclonal Antibody

Product Pathways - Protein Stability

Skp2 (D3G5) XP® Rabbit mAb #2652

Applications Reactivity Sensitivity MW (kDa) Isotype
W IP IHC-P IF-IC H Mk Endogenous 48 Rabbit IgG

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IHC-P=Immunohistochemistry (Paraffin)  IF-IC=Immunofluorescence (Immunocytochemistry)
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

Skp2 (D3G5) XP® Rabbit mAb recognizes endogenous levels of total Skp2 protein. This antibody is predicted to cross-react with Skp2α and Skp2β.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the amino terminus of human Skp2 protein.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using Skp2 (D3G5) XP® Rabbit mAb.

Western Blotting

Western Blotting

Western blot analysis of extracts from 293T cells, either mock transfected (-) or transfected with a Myc/DDK-tagged cDNA expression construct encoding full-length human Skp2 (hSkp2-Myc/DDK, +), using Skp2 (D3G5) XP® Rabbit mAb.

Western Blotting

Western Blotting

Western blot analysis of extracts from HeLa cells expressing either non-targeting shRNA (shNT) or shSkp2, using Skp2 (D3G5) XP® Rabbit mAb (upper) and GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).


IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human lung carcinoma using Skp2 (D3G5) XP® Rabbit mAb.

IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human lymphoma using Skp2 (D3G5) XP® Rabbit mAb.

IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human ovarian carcinoma using Skp2 (D3G5) XP® Rabbit mAb.


IF-IC

IF-IC

Confocal immunofluorescent analyis of HeLa cells expressing either a non-targeting shRNA (shNT; left) or shSkp2 (right) using Skp2 (D3G5) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red) (Cell lines kindly provided by Dr. Wenyi Wei, Harvard Medical School.)

Background

Members of the F-box family of proteins are characterized by the approximate 40 amino acid F-box motif named after cyclin F (1,2). F-box proteins constitute one of the four subunits of the Skp1-Cullin-F-box (SCF) ubiquitin ligase complex. The substrate specificity of SCF complexes is determined by the interchangeable F-box proteins, which act as adaptors by associating with phosphorylated substrate proteins and recruiting them to the SCF core. F-box proteins contain two fundamental domains: the F-box motif mediates binding to Skp1 and a leucine rich repeat (LRR) domain mediates substrate interactions.

S phase kinase-associated protein 2 (Skp2) interacts with cyclin A/CDK2 and mediates proper G1 to S and G2 to M phase transitions by targeting the cyclin-dependent kinase (CDK) inhibitors p27, p21, p130, and the FoxO1 transcription factor for ubiquitination and subsequent proteolysis (3-6). Skp2 protein expression is low in G0 and early G1 phase, increases during late G1 phase, and peaks during S and G2 phases. Inactivation of Skp2 results in S/G2 phase arrested cells with endoduplication and multiple centrosomes (4). Overexpression of Skp2 results in increased CDK activity and contributes to the deregulated proliferation and genetic instabilities typical of cancer cells (7). Research studies have shown that increased Skp2/decreased p27 levels are associated with many aggressive lymphomas and human carcinomas such as colon, breast, prostate and lung cancers (7). Several recent research studies have demonstrated that Skp2 is subject to phosphorylation-dependent regulation by a network of pro-proliferative Ser/Thr kinases. It appears as though phosphorylation of Skp2 at Ser64 by CDK2 (8), Ser72 by Akt1 (9), and Thr417 by PIM1 (10) promotes stabilization of Skp2, possibly constituting an additional mechanism for Skp2 oncogenicity.

  1. Pagano, M. (2004) Mol Cell 14, 414-416.
  2. Reed, S.I. (2003) Nature Rev Mol Cell Biol 4, 855-864.
  3. Zhang, H. et al. (1995) Cell 82, 915-25.
  4. Nakayama, K. et al. (2004) Dev Cell 6, 661-72.
  5. Bornstein, G. et al. (2003) J Biol Chem 278, 25752-7.
  6. Tedesco, D. et al. (2002) Genes Dev 16, 2946-57.
  7. Bloom, J. and Pagano, M. (2003) Semin Cancer Biol 13, 41-7.
  8. Rodier, G. et al. (2008) EMBO J 27, 679-91.
  9. Gao, D. et al. (2009) Nat Cell Biol 11, 397-408.
  10. Cen, B. et al. (2010) J Biol Chem 285, 29128-37.

Application References

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Companion Products

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

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