Revision 1

#14865Store at -20C

Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Applications:

WB, IP

REACTIVITY:

H

SENSITIVITY:

Endogenous

MW (kDa):

48

SOURCE:

Rabbit

UniProt ID:

#Q13309

Entrez-Gene Id:

6502

Product Information

Product Usage Information

Application Dilution
Western Blotting 1:1000
Immunoprecipitation 1:50

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not aliquot the antibody.

Specificity / Sensitivity

Phospho-Skp2 (Ser64) Antibody recognizes endogenous levels of Skp2 protein only when phosphorylated at Ser64. This antibody also cross-reacts with an unidentified 160 kDa phosphoprotein in some cells.

Species Reactivity:

Human

Source / Purification

Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser64 of human Skp2 protein. Antibodies are purified by protein A and peptide affinity chromatography.

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 G1 to S and G2 to M phase transitions by targeting the cyclin-dependent kinase (CDK) inhibitors p27, p21, and p130 for ubiquitination and subsequent proteolysis (3-6). Overexpression of Skp2 results in 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, 414-6.
  2. Reed, S.I. (2003) Nat Rev Mol Cell Biol 4, 855-64.
  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.

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Western Blot Buffer

IMPORTANT: For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

Applications Key

WB: Western Blotting IP: Immunoprecipitation

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

限制使用

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专品专有“专供研究使用”的专专或专似的专专声明, 且未专得美国食品和专品管理局或其他外国或国内专管机专专专任何用途的批准、准专或专可。客专不得将任何专品用于任何专断或治专目的, 或以任何不符合专专声明的方式使用专品。CST 专售或专可的专品提供专作专最专用专的客专,且专用于研专用途。将专品用于专断、专防或治专目的, 或专专售(专独或作专专成)或其他商专目的而专专专品,均需要 CST 的专独专可。客专:(a) 不得专独或与其他材料专合向任何第三方出售、专可、 出借、捐专或以其他方式专专或提供任何专品,或使用专品制造任何商专专品,(b) 不得复制、修改、逆向工程、反专专、 反专专专品或以其他方式专专专专专品的基专专专或技专,或使用专品开专任何与 CST 的专品或服专专争的专品或服专, (c) 不得更改或专除专品上的任何商专、商品名称、徽专、专利或版专声明或专专,(d) 只能根据 CST 的专品专售条款和任何适用文档使用专品, (e) 专遵守客专与专品一起使用的任何第三方专品或服专的任何专可、服专条款或专似专专

Revision 1
#14865

Phospho-Skp2 (Ser64) Antibody

Western Blotting Image 1: Phospho-Skp2 (Ser64) Antibody Expand Image
使用 Phospho-Skp2 (Ser64) Antibody(上图)、Skp2 (D3G5) XP® Rabbit mAb #2652(中图)和 GAPDH (D16H11) XP® Rabbit mAb #5174(下图),对表达非靶向 shRNA (shNT) 或 Skp2 shRNA (shSkp2) 的 Hela 细胞的提取物进行蛋白质印迹分析。
Western Blotting Image 2: Phospho-Skp2 (Ser64) Antibody Expand Image
使用 Phospho-Skp2 (Ser64) Antibody(上图)和 DYKDDDDK Tag Antibody #2368(下图),对转染空载 (-) 或转染表达带有 DDK 标记的全长人 Skp2 野生型蛋白 (hSkp2-WT-DDK; +) 或带有 DDK 标记的全长人 Skp2 S64A 蛋白 (hSkp2-S64A-DDK; +) 表达载体的 293T 细胞的提取物进行蛋白质印迹分析。
Western Blotting Image 3: Phospho-Skp2 (Ser64) Antibody Expand Image
使用 Phospho-Skp2 (Ser64) Antibody 对 293T 和 Hela 细胞的提取物进行蛋白质印迹分析。通过不使用肽(左图)或使用 Skp2 非磷酸肽(中图)和 Skp2 (Ser64) 磷酸肽(右图)进行抗体预孵育来验证该抗体的磷酸特异性。
Immunoprecipitation Image 1: Phospho-Skp2 (Ser64) Antibody Expand Image
使用 Normal Rabbit IgG #2729(泳道 2)或 Phospho-Skp2 (Ser64) Antibody(泳道 3),对 293T 细胞的提取物磷酸化 Skp2 进行免疫沉淀。泳道 1 是 10% 输入对照。使用 Phospho-Skp2 (Ser64) Antibody 进行蛋白质印迹分析。