Phospho-Akt (Ser473) (D9E) XP™

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Product Pathways - PI3K / Akt Signaling

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Phospho-Akt (Ser473) (D9E) XP^® Rabbit mAb #4060

PhosphoSitePlus ^® protein, site, and accession data: Akt1 , Akt2 , Akt3

Applications	Reactivity	Sensitivity	MW (kDa)	Isotype
W IP IHC-P IHC-F IF-IC F	H M R Hm Mk Dm Z B (C) (X) (Dg) (Pg)	Endogenous	60	Rabbit IgG

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 cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Protocols

4060:

Flow , IHC / Frozen* , IHC / Paraffin , Immunofluorescence , Immunoprecipitation , Western Blotting

* Product-specific protocol.

Specificity / Sensitivity

Phospho-Akt (Ser473) (D9E) XP^® Rabbit mAb detects endogenous levels of Akt only when phosphorylated at Ser473.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues around Ser473 of human Akt.

Western Blotting

Western blot analysis of extracts from PC-3 cells, untreated or LY294002/wortmannin-treated, and NIH/3T3 cells, serum-starved or PDGF-treated, using Phospho-Akt (Ser473) (D9E) XP^® Rabbit mAb (upper) or Akt (pan) (C67E7) Rabbit mAb #4691 (lower).

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded MDA-MB-468 xenograft using Phospho-Akt (Ser473) (D9E) XP^® Rabbit mAb (left) or PTEN (138G6) Rabbit mAb #9559 (right). Note the presence of P-Akt staining in the PTEN deficient MDA-MB-468 cells.

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human breast carcinoma comparing SignalStain^® Antibody Diluent #8112 (left) to TBST/5% normal goat serum (right) using Phospho-Akt (Ser473) (D9E) XP^® Rabbit mAb #4060.

 

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Phospho-Akt (Ser473) (D9E) XP^® Rabbit mAb.

IHC-P (paraffin)

Immunohistochemical analysis using Phospho-Akt (Ser473) (D9E) XP^® Rabbit mAb on SignalSlide® Phospho-Akt (Ser473) IHC Controls #8101 (paraffin-embedded LNCaP cells, untreated (left) or LY294002-treated (right).

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human lung carcinoma using Phospho-Akt (Ser473) (D9E) XP^® Rabbit mAb.

 

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded PTEN heterozygous mutant mouse endometrium using Phospho-Akt (Ser473) (D9E) XP^® Rabbit mAb. (Tissue section courtesy of Dr. Sabina Signoretti, Brigham and Women's Hospital, Harvard Medical School, Boston, MA.)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded U-87MG xenograft, untreated (left) or lambda phosphatase-treated (right), using Phospho-Akt (Ser473) (D9E) XP^® Rabbit mAb.

IHC-F (frozen)

Immunohistochemical analysis of frozen SKOV3 xenograft using Phospho-Akt (Ser473) (D9E) XP^® Rabbit mAb.

 

Flow Cytometry

Flow cytometric analysis of Jurkat cells, untreated (green) or treated with LY294002, wortmannin and U0126 (blue), using Phospho-Akt (Ser473) (D9E) XP^® Rabbit mAb compared to a nonspecific negative control antibody (red).

IF-IC

Confocal immunofluorescent analysis of C2C12 cells, LY294002-treated (left) or insulin-treated (right), using Phospho-Akt (Ser473) (D9E) XP^® Rabbit mAb (green). Actin filaments have been labeled with Alexa Fluor^® 555 phalloidin (red). Blue pseudocolor = DRAQ5^® #4084 (fluorescent DNA dye).

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 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 (18,19).

 

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