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Kit Includes

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

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).


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.


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.

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.  Myers, M.P. et al. (1998) Proc Natl Acad Sci USA 95, 13513-8.

2.  Marshall, C.J. (1995) Cell 80, 179-85.

3.  Jefferies, H. B. et al. (1997) EMBO J. 15, 3693-3704.

4.  Cowley, S. et al. (1994) Cell 77, 841-852.

5.  Sarbassov, D.D. et al. (2005) Science 307, 1098-101.

6.  Flotow, H. and Thomas, G. (1992) J Biol Chem 267, 3074-8.

7.  Hunter, T. (1995) Cell 80, 225-36.

8.  Hill, C.S. and Treisman, R. (1995) Cell 80, 199-211.

9.  Sturgill, T.W. et al. (1988) Nature 334, 715-8.

10.  Payne, D.M. et al. (1991) EMBO J 10, 885-92.

11.  Franke, T.F. et al. (1997) Cell 88, 435-7.

12.  Burgering, B.M. and Coffer, P.J. (1995) Nature 376, 599-602.

13.  Owens, D.M. and Keyse, S.M. (2007) Oncogene 26, 3203-13.

14.  Franke, T.F. et al. (1995) Cell 81, 727-36.

15.  Vlahos, C.J. et al. (1994) J Biol Chem 269, 5241-8.

16.  Alessi, D.R. et al. (1996) EMBO J 15, 6541-51.

17.  Peterson, R.T. and Schreiber, S.L. (1998) Curr Biol 8, R248-50.

18.  Ferrari, S. et al. (1991) J Biol Chem 266, 22770-5.

19.  Ma, L. et al. (2005) Cell 121, 179-93.


Entrez-Gene Id 207 , 208 , 10000 , 5595 , 5594 , 6194
Swiss-Prot Acc. P31749 , P31751 , Q9Y243 , P27361 , P28482 , P62753

Protein Specific References

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Schönherr E et al. (2001) J Biol Chem 276, 40687–92

Hill MM et al. (2001) J Biol Chem 276, 25643–6

Rane MJ et al. (2001) J Biol Chem 276, 3517–23

Dhawan P et al. (2002) Cancer Res 62, 7335–42

Conus NM et al. (2002) J Biol Chem 277, 38021–8

Sano H et al. (2002) J Biol Chem 277, 19439–47

Egawa K et al. (2002) J Biol Chem 277, 38863–9

Kisseleva MV et al. (2002) J Biol Chem 277, 6266–72

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Rani MR et al. (2002) J Biol Chem 277, 38456–61

Ho R et al. (2002) Cancer Res 62, 6462–6

Wan X and Helman LJ (2003) Oncogene 22, 8205–11

Fukuda T et al. (2003) J Biol Chem 278, 51324–33

Kim HH et al. (2003) FASEB J 17, 2163–5

Min YH et al. (2004) Cancer Res 64, 5225–31

Tazzari PL et al. (2004) Br J Haematol 126, 675–81

Matsuzaki H et al. (2004) Biochemistry 43, 4284–93

Wolfrum S et al. (2004) Arterioscler Thromb Vasc Biol 24, 1842–7

Kaneko Y et al. (2004) J Cell Sci 117, 407–15

Esfandiarei M et al. (2004) J Virol 78, 4289–98

Baudhuin LM et al. (2004) FASEB J 18, 341–3

Dietze EC et al. (2004) Oncogene 23, 3851–62

Wu T et al. (2004) Mol Cancer Ther 3, 299–307

Honjo S et al. (2005) DNA Cell Biol 24, 141–7

Karlsson HK et al. (2005) Diabetes 54, 1459–67

Viniegra JG et al. (2005) J Biol Chem 280, 4029–36

Le XF et al. (2005) J Biol Chem 280, 2092–104

Smith E and Frenkel B (2005) J Biol Chem 280, 2388–94

Edwards LA et al. (2005) Oncogene 24, 3596–605

Karlsson HK et al. (2005) Diabetes 54, 1692–7

Kippenberger S et al. (2005) J Biol Chem 280, 3060–7

Jung HS et al. (2005) Mol Endocrinol 19, 2748–59

Khundmiri SJ et al. (2006) Am J Physiol Cell Physiol 291, C1247–57

Hers I and (2007) Blood 110, 4243–52

Ananthanarayanan B et al. (2007) J Biol Chem 282, 36634–41

Zunder ER et al. (2008) Cancer Cell 14, 180–92

Grenegård M et al. (2008) J Biol Chem 283, 18493–504

Abubaker J et al. (2009) Mol Cancer 8, 51

Chen PL and Easton AS (2011) Curr Neurovasc Res 8, 14–24

Van Aller GS et al. (2011) Biochem Biophys Res Commun 406, 194–9

Ou YH et al. (2011) Mol Cell 41, 458–70

Uesugi A et al. (2011) Cancer Res 71, 5765–78

Wang S et al. (2012) PLoS One 7, e37427

Glidden EJ et al. (2012) J Biol Chem 287, 581–8

Shih MC et al. (2012) Oncogene 31, 2389–400

Misra UK and Pizzo SV (2012) J Cell Biochem 113, 1488–500

Johnson AL et al. (2001) Biol Reprod 64, 1566–74

Zhang M and Riedel H (2009) J Cell Biochem 107, 65–75

Syme CA et al. (2005) J Biol Chem 280, 11281–8

Li Z et al. (2001) J Biol Chem 276, 42226–32


For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
PathScan is a trademark of Cell Signaling Technology, Inc.

8999
PathScan® Signaling Nodes Multiplex IF Kit