Revision 1
Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Products Included No. Volume Applicaton Dilution Reactivity
Primary Cocktail 8998 100 µl Immunofluorescence (Immunocytochemistry) 1:100 Human Mouse Rat Monkey
Detection Cocktail 8997 100 µl Immunofluorescence (Immunocytochemistry) 1:100 N/A
Kit Analytes Detection Dye Ex(max) (nm) Em(max) (nm)
Phospho-Akt (Ser473) Alexa Fluor® 555 555 565
Phospho-p44/42 Erk1/2 (Thr202/Tyr204) Alexa Fluor® 488 495 519
Phospho-S6 Ribosomal Protein (Ser235/236) Alexa Fluor® 647 650 665
Applications:

IF-IC

:

:

:

:

UniProt ID:

#P27361, #P31751, #Q9Y243, #P62753, #P28482, #P31749

Entrez-Gene Id:

5595, 208, 10000, 6194, 5594, 207

Product Information

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM, NaCl, 100 μg/ml BSA and 50% glycerol. Store at –20°C. Do not aliquot either cocktail.

Specificity / Sensitivity

Phospho-Akt (Ser473) antibody detects endogenous levels of Akt only when phosphorylated at Ser473. Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody detects endogenous levels of p44 and p42 MAP kinase (Erk1 and Erk2) when dually phosphorylated at Thr202 and Tyr204 of Erk1 (Thr185 and Tyr187 of Erk2), and singly phosphorylated at Thr202. This antibody does not cross-react with the corresponding phosphorylated residues of either JNK/SAPK or p38 MAP kinases. Phospho-S6 ribosomal protein (Ser235/236) antibody detects endogenous levels of ribosomal protein S6 only when phosphorylated at Ser235 and Ser236.

Species Reactivity:

Human, Mouse, Rat, Monkey

Source / Purification

Monoclonal antibodies are produced by immunizing animals with synthetic phosphopeptides corresponding to residues surrounding Ser473 of human Akt, Thr202/Tyr204 of human p44 MAP kinase, and Ser235/Ser236 of human ribosomal protein S6.

Product Description

Traditional biochemical and lysate-based assays (e.g., western blot, immunoprecipitation, ELISA) have been integral in the analysis of individual signaling events, however they are limited in their ability to monitor the phosphorylation and subcellular localization of multiple proteins on a per cell basis. PathScan® Signaling Nodes Multiplex IF Kit offers a novel method to simultaneously monitor signaling through key pathway nodes using manual immunofluorescence microscopy, or automated imaging and laser scanning high content platforms. These kits contains a cocktail of three high quality primary antibodies targeted against phospho-Akt (Ser473), phospho-p44/42 (Thr202/Tyr204), and phospho-S6 (Ser235/236) and a detection cocktail utilizing the Alexa Fluor® series of fluorescent dyes. Antibody formulation and dye pairings have been pre-optimized and each kit contains enough reagents for 100 assays (based on a working volume of 100 μL/test).
MW (kDa) n/a

Background

Akt, also referred to as PKB or Rac, plays a critical role in controlling the balance between survival and apoptosis (1-3). This protein kinase is a downstream effector of phosphoinositide-3 kinase (PI3K), and is activated by phospholipid binding and activation loop phosphorylation at Thr308 by PDK1 (4), as well as by phosphorylation within the carboxy terminus at Ser473 by the mTOR-rictor complex (TORC2) (5). This pathway is down-regulated following dephosphorylation of phosphatidyl-inositol 3,4,5 triphosphate by PTEN, as well as by deactivation of PI3K with targeted small molecule inhibitors such as wortmannin and LY294002 (2,3,6,7).
p70 S6 kinase, a mitogen activated Ser/Thr protein kinase downstream of PI3K and the mTOR-raptor complex (mTORC1), phosphorylates the S6 protein of the 40S ribosomal subunit leading to an increase in translation of mRNA transcripts that contain an oligopyrimidine tract in their 5’ untranslated region (8). These particular mRNA transcripts (5’TOP) encode proteins involved in cell cycle progression, as well as ribosomal proteins and elongation factors necessary for translation (8,9). Important S6 ribosomal protein phosphorylation sites include several residues (Ser235, Ser236, Ser240 and Ser244) located within a small, carboxy-terminal region of the S6 protein (10,11).
Both p44 and p42 mitogen-activated protein (MAP) kinases (Erk1 and Erk2, respectively) play a critical role in the regulation of cell growth and differentiation (12-15). MAP kinases are activated by a wide variety of extracellular signals including growth and neurotrophic factors, cytokines, hormones, and neurotransmitters. Activation of MAP kinases occur through phosphorylation of Thr202/Tyr204 on human Erk1 and Thr185/Tyr187 on human Erk2 at the sequence T*EY* by a pair of upstream MAP kinase kinases (MEK1/2) (16,17). Erk proteins are negatively regulated by a family of dual specificity (Thr/Tyr) MAPK phosphatases, known as DUSPs or MKPs (18), along with MEK inhibitors such as U0126 and PD98059. Erk dependent phosphorylation of TSC2 at Ser663 leads to the functional inactivation of the TSC1/TSC2 inhibitory complex, and subsequent downstream activation of S6 ribosomal protein through the mTORC1/p70 S6K signaling cascade (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. Myers, M.P. et al. (1998) Proc Natl Acad Sci U S A 95, 13513-8.
  7. Vlahos, C.J. et al. (1994) J Biol Chem 269, 5241-8.
  8. Peterson, R.T. and Schreiber, S.L. (1998) Curr Biol 8, R248-50.
  9. Jefferies, H.B. et al. (1997) EMBO J 16, 3693-704.
  10. Ferrari, S. et al. (1991) J Biol Chem 266, 22770-5.
  11. Flotow, H. and Thomas, G. (1992) J Biol Chem 267, 3074-8.
  12. Marshall, C.J. (1995) Cell 80, 179-85.
  13. Hunter, T. (1995) Cell 80, 225-36.
  14. Hill, C.S. and Treisman, R. (1995) Cell 80, 199-211.
  15. Cowley, S. et al. (1994) Cell 77, 841-52.
  16. Sturgill, T.W. et al. (1988) Nature 334, 715-8.
  17. Payne, D.M. et al. (1991) EMBO J 10, 885-92.
  18. Owens, D.M. and Keyse, S.M. (2007) Oncogene 26, 3203-13.
  19. Ma, L. et al. (2005) Cell 121, 179-93.

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Applications Key

IF-IC: Immunofluorescence (Immunocytochemistry)

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
PathScan is a trademark of Cell Signaling Technology, Inc.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

Limited Uses

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Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.