Product Pathways - PI3K / Akt Signaling
Phospho-Akt1 (Ser473) (D7F10) XP® Rabbit mAb (Akt1 Specific) #9018
|W IP IF-IC F||H M R||Endogenous||60||Rabbit IgG|
Reactivity Key: H=Human M=Mouse R=Rat
Species cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.
Specificity / Sensitivity
Phospho-Akt1 (Ser473) (D7F10) XP® Rabbit mAb (Akt1 Specific) recognizes endogenous levels of Akt1 protein only when phosphorylated at Ser473. It does not detect Akt2 protein when phosphorylated at Ser474.
Source / Purification
Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser473 of human Akt1 protein.
Western blot analysis of extracts from LNCaP cells, transfected with a construct expressing Akt1 shRNA or Akt2 shRNA, uninduced (-) or doxycycline-induced for the indicated times, using Phospho-Akt1 (Ser473) (D7F10) XP® Rabbit mAb (Akt1 Specific) (top), Phospho-Akt2 (Ser474) (D3H2) Rabbit mAb (Akt2 Specific) #8599 (2nd from top), Akt1 (C73H10) Rabbit mAb #2938 (middle), Akt2 (D6G4) Rabbit mAb #3063 (2nd from bottom), or PI3 Kinase p85 (19H8) Rabbit mAb #4257 (bottom). (Data kindly provided by Drs. Rebecca Chin and Alex Toker, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA).
Western blot analysis of purified recombinant phospho-Akt1, phospho-Akt2 and phospho-Akt3 proteins using Phospho-Akt1 (Ser473) (D7F10) XP® Rabbit mAb (Akt1 Specific) (upper), Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb #4060 (middle) and Akt (pan) (C67E7) Rabbit mAb #4691 (lower).
Western blot analysis of extracts from Akt1 (-/-) mouse embryonic fibroblast (MEF) or Akt2 (-/-) MEF, untreated or stimulated with hPDGF #8913 (100 ng/ml, 15 min), using Phospho-Akt1 (Ser473) (D7F10) XP® Rabbit mAb (Akt1 Specific) (upper), Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb #4060 (middle), or Akt (pan) (C67E7) Rabbit mAb #4691 (lower).
Flow cytometric analysis of Akt1 (-/-) mouse embryonic fibroblast (MEF) cells (blue) or Akt2 (-/-) MEF cells (green), PDGF-treated (100 ng/mL, 15 min), using Phospho-Akt1 (Ser473) (D7F10) XP® Rabbit mAb (Akt1 Specific). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary Ab.
Confocal immunofluorescent analysis of Akt1 (-/-) mouse embryonic fibroblast (MEF) (left column), or Akt2 (-/-) MEF (right column) cells, stimulated with hPDGF #8913 (100 ng/ml, 15 min) (top row) or inhibited with LY294002 #9901 (bottom row), using Phospho-Akt1 (Ser473) (D7F10) XP® Rabbit mAb (Akt1 Specific) (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
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).
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- 8599 Phospho-Akt2 (Ser474) (D3H2) Rabbit mAb (Akt2 Specific)
- 4060 Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb
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- 7003 20X LumiGLO® Reagent and 20X Peroxide
- 9997 Tris Buffered Saline with Tween 20 (TBST-10X)
- 9998 BSA
- 9999 Nonfat Dry Milk
For Research Use Only. Not For Use In Diagnostic Procedures.