Product Pathways - Akt Signaling
Phospho-Akt Pathway Sampler Kit #9916
| Kit Includes | Quantity | Applications | Reactivity | MW (kDa) | Source |
|---|---|---|---|---|---|
| Phospho-Akt (Ser473) (D9E) Rabbit mAb # 4060 | 40 microliters | W IP IHC-P IF-IC F | H M R Dr Z | 60 | Rabbit |
| Phospho-Akt (Thr308) (C31E5E) Rabbit mAb # 2965 | 40 microliters | W IF-IC F | H M R Mk | 60 | Rabbit |
| Akt (pan) (C67E7) Rabbit mAb # 4691 | 40 microliters | W IP IHC-P IF-IC F | H M R Mk | 60 | Rabbit |
| Phospho-c-Raf (Ser259) Antibody # 9421 | 40 microliters | W IP | H M R X (C) | 74 | Rabbit |
| Phospho-GSK-3β (Ser9) Antibody # 9336 | 40 microliters | W | H M R Mk (Z) | 46 | Rabbit |
| Phospho-PTEN (Ser380) Antibody # 9551 | 40 microliters | W IP | H M R | 54 | Rabbit |
| Phospho-PDK1 (Ser241) Antibody # 3061 | 40 microliters | W IP | H M R | 58 to 68 | Rabbit |
| Anti-rabbit IgG, HRP-linked Antibody # 7074 | 100 microliters | Goat | |||
| LY294002 (PI3 Kinase Inhibitor) # 9901 | 0.3 milligrams | 307 |
Applications Key:
W=Western Blotting
IP=Immunoprecipitation
IHC-P=Immunohistochemistry (Paraffin)
IF-IC=Immunofluorescence (Immunocytochemistry)
F=Flow Cytometry
Reactivity Key:
H=Human
M=Mouse
R=Rat
Mk=Monkey
C=Chicken
Dr=Drosophila
X=Xenopus
Z=Zebra Fish
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 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 blot analysis of extracts from NIH/3T3 cells (starved for 16 hours) treated with PDGF (50 ng/ml), using Phospho-PDK1 (Ser241) Antibody #3061 (upper) or control PDK1 antibody #3062 (lower). The phospho-specificity of the antibody was confirmed by treating the membrane with calf intestinal alkaline phosphatase (CIP) after Western transfer.
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) Rabbit mAb #4060.
Western Blotting
Western blot analysis of extracts from PDGF treated NIH/3T3 cells, using Phospho-GSK-3beta (Ser9) Antibody #9336 or control GSK-3beta Antibody #9332.
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 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.
Source / Purification
Antibodies are produced by immunizing rabbits with synthetic phospho-peptides corresponding to residues surrounding Ser473 or Thr308 of mouse Akt, Ser9 of human GSK-3beta, 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 by phosphorylating and inactivating 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 Kip (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). Inhibition of mTOR stops the protein synthesis machinery due to inactivation of its effector, p70 S6 kinase and activation of the eukaryotic initiation factor 4E binding protein 1 (4E-EP1), an inhibitor of translation (18,19).
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- Franke, T.F. et al. (1995) Cell 81, 727-36.
- Alessi, D.R. et al. (1996) EMBO J 15, 6541-51.
- Sarbassov, D.D. et al. (2005) Science 307, 1098-101.
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- Cardone, M.H. et al. (1998) Science 282, 1318-21.
- Brunet, A. et al. (1999) Cell 96, 857-68.
- Zimmermann, S. and Moelling, K. (1999) Science 286, 1741-4.
- Cantley, L.C. and Neel, B.G. (1999) Proc Natl Acad Sci USA 96, 4240-5.
- Vlahos, C.J. et al. (1994) J Biol Chem 269, 5241-8.
- Hajduch, E. et al. (2001) FEBS Lett 492, 199-203.
- Cross, D.A. et al. (1995) Nature 378, 785-9.
- Diehl, J.A. et al. (1998) Genes Dev 12, 3499-511.
- Gesbert, F. et al. (2000) J Biol Chem 275, 39223-30.
- Zhou, B.P. et al. (2001) Nat Cell Biol 3, 245-52.
- Nave, B.T. et al. (1999) Biochem J 344 Pt 2, 427-31.
- Inoki, K. et al. (2002) Nat Cell Biol 4, 648-57.
- Manning, B.D. et al. (2002) Mol Cell 10, 151-62.
Application References
- Crowder, R.J. and Freeman, R.S. (2000) J Biol Chem 275, 34266-71. This article references the use of Phospho-Akt Pathway Sampler Kit in the following applications: Western Blotting
- Guo, M. et al. (2000) Biochem Pharmacol 60, 635-42. This article references the use of Phospho-Akt Pathway Sampler Kit in the following applications: PI3K inhibition
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