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9916
Phospho-Akt Pathway Antibody Sampler Kit

Phospho-Akt Pathway Antibody Sampler Kit #9916

Western Blotting Image 1

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).

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Western Blotting Image 2

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

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Western Blotting Image 3

Site specificity of Phospho-c-Raf (Ser259) Antibody: Western blot analysis of recombinant Myc-tagged c-Raf protein, wild-type (lanes 1 and 3) and S259A mutant (lanes 2 and 4), using Phospho-Raf (Ser259) Antibody or a Myc antibody. (Provided by Dr. Guri Tzivion, Massachusetts General Hospital.)

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Western Blotting Image 4

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 (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).

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Western Blotting Image 5

Western blot analysis of extracts from various cell lines, using Phospho-PTEN (Ser380) Antibody (upper) or PTEN Antibody #9552 (lower). The phospho-specificity of the antibody was confirmed by treating the membrane with calf intestinal alkaline phosphatase (CIP) after Western transfer.

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Western Blotting Image 6

Western blot analysis of extracts from PC3 cells, HCT116 wild-type and HCT116 PDK1 -/- cells using Phospho-PDK1 (Ser241) (C49H2) Rabbit mAb (upper) and Akt (pan) (C67E7) Rabbit mAb #4691 (lower). (HCT116 wild-type and HCT116 PDK1 -/- cells were kindly provided by Dr. Bert Vogelstein, Johns Hopkins University, Baltimore, MD).

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Western Blotting Image 7

Western blot analysis of extracts from NIH/3T3 cells, untreated (-) or treated with Human Platelet-Derived Growth Factor AA (hPDGF-AA) #8913 (100 ng/ml, 5 min; +), and untreated (-) LNCaP and PC-3 cells, using Phospho-Akt (Thr308) (D25E6) XP® Rabbit mAb (upper) or Akt (pan) (C67E7) Rabbit mAb #4691 (lower).

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Western Blotting Image 8

After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.

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IHC-P (paraffin) Image 9

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.

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IHC-P (paraffin) Image 10

Immunohistochemical analysis of paraffin-embedded human melanoma using Akt (pan) (C67E7) Rabbit mAb.

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Western Blotting Image 11

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

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Western Blotting Image 12

Western blot analysis of extracts from PC-3 cells, untreated or LY294002/wortmannin-treated, using Phospho-GSK-3β (Ser9) (D85E12) XP® Rabbit mAb (upper) or GSK-3β (27C10) Rabbit mAb #9315 (lower).

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Western Blotting Image 13

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

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IP Image 14

Immunoprecipitation of phospho-Akt (Thr308) from Jurkat cell extracts using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or Phospho-Akt (Thr308) (D25E6) XP® Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using Phospho-Akt (Thr308) (D25E6) XP® Rabbit mAb.

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IHC-P (paraffin) Image 15

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.

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IHC-P (paraffin) Image 16

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).

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Flow Cytometry Image 17

Flow cytometric analysis of NIH/3T3 cells, untreated (blue) or PDGF-treated (green), using Phospho-GSK-3β (Ser9) (D85E12) XP® Rabbit mAb.

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Flow Cytometry Image 18

Flow cytometric analysis of serum-starved NIH/3T3 cells, untreated (blue) or treated with Human Platelet-Derived Growth Factor AA (hPDGF-AA) #8913 (100 ng/ml, 15 min; green), using Phospho-Akt (Thr308) (D25E6) XP® Rabbit mAb. Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 647 Conjugate) #4414 was used as a secondary antibody.

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IHC-P (paraffin) Image 19

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

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IHC-P (paraffin) Image 20

Immunohistochemical analysis 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)).

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IF-IC Image 21

Confocal immunofluorescent analysis of wild type mouse embryonic fibroblasts (MEFs) (top row), GSK-3β (-/-) MEFs (middle row) , or PC-3 cells (bottom row), untreated (left), LY294002- and Wortmannin-treated (#9901 and #9951 respectively; center) or lambda phosphatase-treated (right), using Phospho-GSK-3β (Ser9) (D85E12) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye). (MEF wild type and GSK-3β (-/-) cells were kindly provided by Dr. Jim Woodgett, University of Toronto, Canada).

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IF-IC Image 22

Confocal immunofluorescent analysis of C2C12 cells, insulin-treated (100 nM, 15 min; left) or treated with LY294002 #9901 (50 μM, 2 hr; right), using Phospho-Akt (Thr308) (D25E6) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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IHC-P (paraffin) Image 23

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)).

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Flow Cytometry Image 24

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

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IHC-P (paraffin) Image 25

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

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IF-IC Image 26

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).

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IHC-P (paraffin) Image 27

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.)

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IHC-P (paraffin) Image 28

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

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IHC-F (frozen) Image 29

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

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Flow Cytometry Image 30

Flow cytometric analysis of Jurkat cells, untreated (green) or treated with LY294002 #9901, wortmannin #9951 and U0126 #9903 (blue), using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb compared to a nonspecific negative control antibody (red).

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IF-IC Image 31

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 #8953 (red). Blue pseudocolor = DRAQ5®#4084 (fluorescent DNA dye).

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Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb 4060 20 µl
  • WB
  • IP
  • IHC
  • IF
  • F
H M R Hm Mk Dm Z B 60 Rabbit IgG
Akt (pan) (C67E7) Rabbit mAb 4691 20 µl
  • WB
  • IP
  • IHC
  • IF
  • F
H M R Mk Dm 60 Rabbit IgG
Phospho-c-Raf (Ser259) Antibody 9421 20 µl
  • WB
  • IP
H M R Mk X 74 Rabbit 
Phospho-GSK-3β (Ser9) (D85E12) XP® Rabbit mAb 5558 20 µl
  • WB
  • IP
  • IF
  • F
H M R Hm 46 Rabbit IgG
Phospho-PTEN (Ser380) Antibody 9551 20 µl
  • WB
  • IP
H M R 54 Rabbit 
Phospho-PDK1 (Ser241) (C49H2) Rabbit mAb 3438 20 µl
  • WB
  • IP
H M R 58 to 68 Rabbit IgG
Phospho-Akt (Thr308) (D25E6) XP® Rabbit mAb 13038 20 µl
  • WB
  • IP
  • IF
  • F
H M R Mk 60 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

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 two Western blot experiments.

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

Antibodies are produced by immunizing rabbits with synthetic phosphopeptides corresponding to residues surrounding Ser473 or Thr308 of human Akt, Ser9 of human GSK-3ß, 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.

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.
Entrez-Gene Id
207 , 208 , 10000 , 5894 , 2932 , 5170 , 5728
Swiss-Prot Acc.
P31749 , P31751 , Q9Y243 , P04049 , P49841 , O15530 , P60484
For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.

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