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59633
AS160 Signaling Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit

AS160 Signaling Antibody Sampler Kit #59633

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AS160 Signaling Antibody Sampler Kit: Image 1

Western blot analysis of extracts from HepG2, RD and 293T cells using AS160 (C69A7) Rabbit mAb.

AS160 Signaling Antibody Sampler Kit: Image 2

Western blot analysis of extracts from serum-starved HeLa cells, untreated or treated with either insulin (150 nM, 15 min) or hIGF-I #8917 (100 ng/ml, 15 min), using Phospho-AS160 (Ser318) (D3D11) Rabbit mAb (upper) or AS160 (C69A7) Rabbit mAb #2670 (lower). The phospho-specificity of the antibody is verified by λ phosphatase treatment.

AS160 Signaling Antibody Sampler Kit: Image 3

Western blot analysis of extracts from serum-starved HeLa cells, untreated or treated with hIGF-I #8917 (100 ng/ml, 15 min), using Phospho-AS160 (Ser588) (D8E4) Rabbit mAb (upper) or AS160 (C69A7) Rabbit mAb #2670 (lower). The phospho-specificity of the antibody is verified by λ phosphatase treatment.

AS160 Signaling Antibody Sampler Kit: Image 4

Western blot analysis of extracts from serum starved HeLa cells, untreated (-) or insulin-treated (150 nM, 15 min), using Phospho-AS160 (Thr642) (D27E6) Rabbit mAb (upper) or AS160 (C69A7) Rabbit mAb #2670 (lower). The phospho-specificity of the antibody is verified by λ phosphatase treatment.

AS160 Signaling Antibody Sampler Kit: Image 5

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

AS160 Signaling Antibody Sampler Kit: Image 6

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 (solid line) compared to a concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed line). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.

AS160 Signaling Antibody Sampler Kit: Image 7

Flow cytometric analysis of Jurkat cells, untreated (green) or treated with LY294002 #9901, Wortmannin #9951, and U0126 #9903 (50 μM, 1 μM, and 10 μM, 2 hr; blue) using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.

AS160 Signaling Antibody Sampler Kit: 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.

AS160 Signaling Antibody Sampler Kit: Image 9

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).

AS160 Signaling Antibody Sampler Kit: Image 10

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).

AS160 Signaling Antibody Sampler Kit: Image 11

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).

AS160 Signaling Antibody Sampler Kit: Image 12

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

AS160 Signaling Antibody Sampler Kit: Image 13

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.

AS160 Signaling Antibody Sampler Kit: Image 14

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.

AS160 Signaling Antibody Sampler Kit: Image 15

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).

AS160 Signaling Antibody Sampler Kit: Image 16

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).

AS160 Signaling Antibody Sampler Kit: Image 17

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.

AS160 Signaling Antibody Sampler Kit: Image 18

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)).

AS160 Signaling Antibody Sampler Kit: Image 19

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

AS160 Signaling Antibody Sampler Kit: Image 20

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

AS160 Signaling Antibody Sampler Kit: Image 21

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)).

AS160 Signaling Antibody Sampler Kit: Image 22

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

AS160 Signaling Antibody Sampler Kit: Image 23

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.)

AS160 Signaling Antibody Sampler Kit: Image 24

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

AS160 Signaling Antibody Sampler Kit: Image 25

Immunoprecipitation of phospho-Akt (Ser473) from Jurkat extracts treated with Calyculin A #9902 (100nM, 30 min). Lane 1 is 10% input, lane 2 is Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb, and lane 3 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900. Western blot analysis was performed with Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as a secondary antibody.

AS160 Signaling Antibody Sampler Kit: Image 26

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).

To Purchase # 59633T
Product # Size Price Inventory
59633T
1 Kit  (7 x 20 µl)

Product Description

The AS160 Signaling Antibody Sampler Kit provides an economical means of detecting select components involved in the AS160 signaling pathway. The kit contains enough primary antibodies to perform at least two western blot experiments per antibody.

Specificity / Sensitivity

AS160 (C69A7) Rabbit mAb detects endogenous levels of total AS160 protein. Phospho-AS160 (Ser318) (D3D11) Rabbit mAb recognizes endogenous levels of AS160 protein only when phosphorylated at Ser318. Phospho-AS160 (Ser588) (D8E4) Rabbit mAb recognizes endogenous levels of AS160 protein only when phosphorylated at Ser588. Phospho-AS160 (Thr642) (D27E6) Rabbit mAb recognizes endogenous levels of AS160 protein only when phosphorylated at Thr642. Akt (pan) (C67E7) Rabbit mAb detects endogenous levels of total Akt protein. This antibody does not cross-react with other related proteins. Phospho-Akt (Thr308) (D25E6) XP® Rabbit mAb recognizes endogenous levels of Akt1 protein only when phosphorylated at Thr308. This antibody also recognizes endogenous levels of Akt2 protein when phosphorylated at Thr309 or Akt3 protein when phosphorylated at Thr305. 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 peptide corresponding to residues surrounding Ala195 of human AS160. Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser318 of human AS160 protein. Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser588 of human AS160 protein. Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr642 of human AS160 protein. Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues in the carboxy-terminal sequence of mouse Akt. Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Thr308 of human Akt1 protein. Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues around Ser473 of human Akt.

Background

Insulin is a major hormone controlling critical energy functions, such as glucose and lipid metabolism. Insulin binds to and activates the insulin receptor (IR) tyrosine kinase, which phosphorylates and recruits adaptor proteins. The signaling pathway initiated by insulin and its receptor stimulates glucose uptake in muscle cells and adipocytes through translocation of the Glut4 glucose transporter from the cytoplasm to the plasma membrane (1). A 160 kDa substrate of the Akt Ser/Thr kinase (AS160, TBC1D4) is a Rab GTPase-activating protein that regulates insulin-stimulated Glut4 trafficking. AS160 is expressed in many tissues including brain, kidney, liver, and brown and white fat (2). Multiple Akt phosphorylation sites have been identified on AS160 in vivo, with five sites (Ser318, Ser570, Ser588, Thr642, and Thr751) showing increased phosphorylation following insulin treatment (2,3). Studies using recombinant AS160 demonstrate that insulin-stimulated phosphorylation of AS160 is a crucial step in Glut4 translocation (3) and is reduced in some patients with type 2 diabetes (4). The interaction of 14-3-3 regulatory proteins with AS160 phosphorylated at Thr642 is a necessary step for Glut4 translocation (5). Phosphorylation of AS160 by AMPK is involved in the regulation of contraction-stimulated Glut4 translocation (6).

Akt, also referred to as PKB or Rac, plays a critical role in controlling survival and apoptosis (7-9). This protein kinase is activated by insulin and various growth and survival factors to function in a wortmannin-sensitive pathway involving PI3 kinase (8,9). Akt is activated by phospholipid binding and activation loop phosphorylation at Thr308 by PDK1 (10) and by phosphorylation within the carboxy terminus at Ser473. The previously elusive PDK2 responsible for phosphorylation of Akt at Ser473 has been identified as the mammalian target of rapamycin (mTOR) in a rapamycin-insensitive complex with rictor and Sin1 (11,12).

  1. Watson, R.T. and Pessin, J.E. (2006) Trends Biochem. Sci. 31, 215-22.
  2. Kane, S. et al. (2002) J. Biol. Chem. 277, 22115-8.
  3. Sano, H. et al. (2003) J. Biol. Chem. 278, 14599-602.
  4. Karlsson HK et al. (2005) Diabetes 54, 1692–7
  5. Ramm, G. et al. (2006) J. Biol. Chem. 281, 29174-80.
  6. Kramer, H.F. et al. (2006) J. Biol. Chem. 281, 31478-85.
  7. Franke, T.F. et al. (1997) Cell 88, 435-7.
  8. Burgering, B.M. and Coffer, P.J. (1995) Nature 376, 599-602.
  9. Franke, T.F. et al. (1995) Cell 81, 727-36.
  10. Alessi, D.R. et al. (1996) EMBO J 15, 6541-51.
  11. Sarbassov, D.D. et al. (2005) Science 307, 1098-101.
  12. Jacinto, E. et al. (2006) Cell 127, 125-37.

Pathways & Proteins

Explore pathways + proteins related to this product.

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U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.