Cell Signaling Technology

Product Pathways - Akt Signaling

Akt Isoform Sampler Kit #9940

Kit Includes Quantity Applications Reactivity MW (kDa) Source
Akt1 (C73H10) Rabbit mAb # 2938 40 microliters W IP IHC-P H M R Mk 60 Rabbit
Akt2 (5B5) Rabbit mAb # 2964 40 microliters W IP H M R Mk 60 Rabbit
Akt3 (62A8) Rabbit mAb # 3788 40 microliters W IP H M R 60 Rabbit
Akt (pan) (C67E7) Rabbit mAb # 4691 40 microliters W IP IHC-P IF-IC F H M R Mk 60 Rabbit
Jurkat cell extracts - Untreated 100 microliters
Anti-rabbit IgG, HRP-linked Antibody # 7074 50 microliters Goat

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

Specificity / Sensitivity

Each isoform-specific antibody recognizes endogenous levels of the specified Akt isoform independent of its phosphorylation state. Neither antibody cross-reacts with the two other isoforms. Akt Antibody recognizes endogenous levels of all three Akt isoforms.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell types using Akt1 (C73H10) Rabbit mAb #2938.

Western Blotting

Western Blotting

Western blot analysis of extracts from Akt1 (lane 1), Akt2 (lane 2) and Akt3 (lane 3) knock-out mouse embryonic fibroblasts (MEFs) and matched wild-type MEFs (lane 4) using Akt1 (C73H10) Rabbit mAb #2938 (upper) and Akt2 Antibody #2962 (lower).

Western Blotting

Western Blotting

Western blot analysis of Akt1, Akt2, and Akt3 recombinant proteins, and extracts from Jurkat, C2C12, PC12, and COS cells, using Akt2 (5B5) Rabbit Monoclonal Antibody #2964.


Western Blotting

Western Blotting

Western blot analysis of recombinant Akt1, Akt2 and Akt3 proteins, and extracts from MDA-MB-231 (human), CAD (mouse) and PC12 (rat) cells using Akt3 (62A8) Rabbit mAb #3788 (upper) or Akt Antibody #9272 (lower). Recombinant Akt1, Akt2 and Akt3 are 6His-fusion proteins, MW=66 kDa.

Western Blotting

Western Blotting

Western blot analysis of recombinant Akt1, Akt2 and Akt3 proteins, and extracts from various cell lines, using Akt (pan) (C67E7) Rabbit mAb #4691.

Western Blotting

Western Blotting

Western blot analysis of recombinant Akt1, Akt2 and Akt3 proteins, and extracts from various cell lines, using Akt (pan) (C67E7) Rabbit mAb #4691.


Western Blotting

Western Blotting

Western blot analysis of recombinant Akt1, Akt2 and Akt3 proteins, and extracts from MDA-MB-231 (human), CAD (mouse) and PC12 (rat) cells, using Akt3 (62A8) Rabbit mAb (upper) or Akt Antibody #9272 (lower). Recombinant Akt1, Akt2 and Akt3 are 6His-fusion proteins, MW=66 kDa.

IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis using Akt1 (C73H10) Rabbit mAb on SignalSlide™ Phospho-Akt (Ser473) IHC Controls #8101 (paraffin-embedded LNCaP cells, untreated (left) or LY294002-treated (right)).

IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Akt1 Rabbit mAb in the presence of control peptide (left) or antigen specific peptide (right).


IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Akt (pan) (C67E7) Rabbit mAb in the presence of control peptide (left) or Akt (pan) Blocking Peptide #1085 (right).

IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of using Akt (pan) (C67E7) Rabbit mAb on SignalSlide (TM) Phospho-Akt (Ser473) IHC Controls #8101 (paraffin-embedded LNCaP cells, untreated (left) or LY294002-treated (right)).

Flow Cytometry

Flow Cytometry

Flow cytometric analysis of Jurkat cells using Akt (pan) (C67E7) Rabbit mAb (blue) compared to a nonspecific negative control antibody (red).


IF-IC

IF-IC

Confocal immunofluorescent analysis of C2C12 cells, LY294002-treated (left) or insulin-treated (right), using Akt (pan) (C67E7) Rabbit mAb (green). Actin filaments have been labeled with Alexa Fluor® 555 phalloidin (red). Blue pseudocolor = DRAQ5™ (fluorescent DNA dye).

Source / Purification

Each isoform-specific antibody is produced by immunizing mice or rabbits with a synthetic peptide (KLH-coupled) that is specific for the Akt isoform. Akt1 Monoclonal antibody is produced by immunizing rabbits with a synthetic peptide (KLH-coupled) surrounding Leu110 of human Akt1. Akt2 Monoclonal antibody #2964 is produced by immunizing rabbits with a synthetic peptide (KLH-coupled) corresponding to residues of human Akt2. Akt3 Monoclonal antibody is produced by immunizing rabbits with a synthetic peptide (KLH-coupled) corresponding to residues of human Akt3. Akt(pan) Monoclonal antibody is produced by immunizing rabbits with a synthetic peptide (KLH-coupled) at the carboxy-terminal sequence of mouse Akt.

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).

  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. Nave, 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.

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