Cell Signaling Technology

Product Pathways - PI3K / Akt Signaling

Phospho-Akt Pathway Antibody Sampler Kit #9916

Kit Includes Quantity Applications Reactivity MW (kDa) Isotype
Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb #4060 40 µl W IP IHC-P IHC-F IF-IC F H M R Hm Mk Dm Z B (C) (X) (Dg) (Pg) 60 Rabbit IgG
Phospho-Akt (Thr308) (C31E5E) Rabbit mAb #2965 40 µl W IF-IC F H M R Hm Mk 60 Rabbit IgG
Akt (pan) (C67E7) Rabbit mAb #4691 40 µl W IP IHC-P IF-IC F H M R Mk Dm (Pg) 60 Rabbit IgG
Phospho-c-Raf (Ser259) Antibody #9421 40 µl W IP H M R X (C) 74 Rabbit
Phospho-GSK-3β (Ser9) (D85E12) XP® Rabbit mAb #5558 40 µl W IP IF-IC F H M R Hm 46 Rabbit IgG
Phospho-PTEN (Ser380) Antibody #9551 40 µl W IP H M R (C) 54 Rabbit
Phospho-PDK1 (Ser241) (C49H2) Rabbit mAb #3438 40 µl W IP H M R 58 to 68 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody #7074 100 µl Goat
LY294002 #9901 0.3 milligrams 307.34

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IHC-P=Immunohistochemistry (Paraffin)  IHC-F=Immunohistochemistry (Frozen)  IF-IC=Immunofluorescence (Immunocytochemistry)  F=Flow Cytometry
Reactivity Key:  H=Human  M=Mouse  R=Rat  Hm=Hamster  Mk=Monkey  C=Chicken  Dm=D. melanogaster  X=Xenopus  Z=Zebrafish  B=Bovine  Dg=Dog  Pg=Pig
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Specificity / Sensitivity

Each phospho-specific antibody recognizes the phosphorylated form of its target. Akt Antibody recognizes total Akt protein, independent of its phosphorylation state.

Western Blotting

Western Blotting

Western blot analysis of extracts from NIH/3T3 and Jurkat cells, untreated, PDGF-treated or LY294002-treated as indicated, using Phospho-Akt (Thr308) (C31E5) Rabbit mAb #2965 (upper) or Akt (pan) (C67E7) Rabbit mAb #4691 (lower).

Western Blotting

Western Blotting

Western blot analysis of extracts from PC3 cells, untreated or λ phosphatase-treated, using Phospho-PDK1 (Ser241) (C49H2) Rabbit mAb #3438 (upper), PDK1 Antibody #3062 (middle) or Akt Antibody #9272 (lower).

Western Blotting

Western Blotting

Western blot analysis of extracts from PC3 cells, untreated or LY294002/wortmannin-treated, and NIH/3T3 cells, serum- starved or PDGF-treated, using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb #4060.


Western Blotting

Western Blotting

Western blot analysis of extracts from GSK-3α (-/-) (lanes 1,2) and GSK-3β (-/-) (lanes 3,4) mouse embryonic fibroblast (MEF) cells, λ phosphatase or PDGF-treated, using Phospho-GSK-3β (Ser9) (D85E12) XP® Rabbit mAb #5558 (upper) and α/β-Tubulin Antibody #2148 (lower). (MEF wild type, GSK-3α (-/-) and GSK-3β (-/-) cells were kindly provided by Dr. Jim Woodgett, University of Toronto, Canada).

Western Blotting

Western Blotting

Western blot analysis of extracts from wild type (lanes 1,2), GSK-3α (-/-) (lanes 3,4) and GSK-3β (-/-) (lanes 5,6) mouse embryonic fibroblast cells (MEF), untreated or PDGF treated, using Phospho-GSK-3β (Ser9) (5B3) Rabbit mAb #9323 (upper) and GSK-3α/β Antibody (lower). (MEF wild type, GSK-3α (-/-) and GSK-3β (-/-) cells were kindly provided by Dr. Jim Woodgett, University of Toronto, Canada).

Western Blotting

Western Blotting

Western blot analysis of extracts from HeLa cells, untreated or TPA-treated, using Phospho-Raf (Ser259) Antibody #9421 (upper), or a total c-Raf antibody (lower).


Western Blotting

Western Blotting

Western blot analysis of various cell lines with Phospho-PTEN (Ser380) Antibody #9551 (upper) or PTEN Antibody #9552 (lower). The phospho-specificity of the antibody was characterized by treating the membrane without (-) or with (+) calf intestinal alkaline phosphatase (CIP) after Western transfer.

Description

The Phospho-Akt Pathway Antibody Sampler Kit provides an economical means to evaluate the activation status of the Akt signaling pathway, including PTEN and phosphorylated Akt, GSK-3beta, c-Raf and PDK1. The kit includes enough primary and secondary antibodies to perform four Western blot experiments, as well as a specific inhibitor of PI3 kinase (LY294002).

Source / Purification

Antibodies are produced by immunizing rabbits with synthetic phosphopeptides corresponding to residues surrounding Ser473 or Thr308 of mouse Akt, Ser9 of human GSK-3β, Ser259 of human c-Raf, Ser380 of human PTEN or Ser241 of human PDK1. Polyclonal antibodies are purified by protein A and peptide affinity chromatography.

Background

Akt, also referred to as PKB or Rac, plays a critical role in controlling survival and apoptosis (1-3). This protein kinase is activated by insulin and various growth and survival factors to function in a wortmannin-sensitive pathway involving PI3 kinase (2,3). Akt is activated by phospholipid binding and activation loop phosphorylation at Thr308 by PDK1 (4) and by phosphorylation within the carboxy terminus at Ser473. The previously elusive PDK2 responsible for phosphorylation of Akt at Ser473 has been identified as mammalian target of rapamycin (mTOR) in a rapamycin-insensitive complex with rictor and Sin1 (5,6). Akt promotes cell survival by inhibiting apoptosis through phosphorylation and inactivation of several targets, including Bad (7), forkhead transcription factors (8), c-Raf (9), and caspase-9. PTEN phosphatase is a major negative regulator of the PI3 kinase/Akt signaling pathway (10). LY294002 is a specific PI3 kinase inhibitor (11). Another essential Akt function is the regulation of glycogen synthesis through phosphorylation and inactivation of GSK-3α and β (12,13). Akt may also play a role in insulin stimulation of glucose transport (12). In addition to its role in survival and glycogen synthesis, Akt is involved in cell cycle regulation by preventing GSK-3β-mediated phosphorylation and degradation of cyclin D1 (14) and by negatively regulating the cyclin dependent kinase inhibitors p27 Kip1 (15) and p21 Waf1/Cip1 (16). Akt also plays a critical role in cell growth by directly phosphorylating mTOR in a rapamycin-sensitive complex containing raptor (17). More importantly, Akt phosphorylates and inactivates tuberin (TSC2), an inhibitor of mTOR within the mTOR-raptor complex (18,19).

  1. Franke, T.F. et al. (1997) Cell 88, 435-7.
  2. Burgering, B.M. and Coffer, P.J. (1995) Nature 376, 599-602.
  3. Franke, T.F. et al. (1995) Cell 81, 727-36.
  4. Alessi, D.R. et al. (1996) EMBO J 15, 6541-51.
  5. Sarbassov, D.D. et al. (2005) Science 307, 1098-101.
  6. Jacinto, E. et al. (2006) Cell 127, 125-37.
  7. Cardone, M.H. et al. (1998) Science 282, 1318-21.
  8. Brunet, A. et al. (1999) Cell 96, 857-68.
  9. Zimmermann, S. and Moelling, K. (1999) Science 286, 1741-4.
  10. Cantley, L.C. and Neel, B.G. (1999) Proc Natl Acad Sci USA 96, 4240-5.
  11. Vlahos, C.J. et al. (1994) J Biol Chem 269, 5241-8.
  12. Hajduch, E. et al. (2001) FEBS Lett 492, 199-203.
  13. Cross, D.A. et al. (1995) Nature 378, 785-9.
  14. Diehl, J.A. et al. (1998) Genes Dev 12, 3499-511.
  15. Gesbert, F. et al. (2000) J Biol Chem 275, 39223-30.
  16. Zhou, B.P. et al. (2001) Nat Cell Biol 3, 245-52.
  17. Navé, B.T. et al. (1999) Biochem J 344 Pt 2, 427-31.
  18. Inoki, K. et al. (2002) Nat Cell Biol 4, 648-57.
  19. Manning, B.D. et al. (2002) Mol Cell 10, 151-62.

Application References

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Protocols

* Product-specific protocol.

Companion Products

Selected rabbit monoclonal antibodies are produced under license (granting certain rights including those under U. S. Patent No. 5,675,063 and/or U.S.S.N. 11/476,277) from Epitomics, Inc.

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

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