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Analysis of A549 cells exposed to varying concentrations of LY294002 (PI3 Kinase Inhibitor) #9901 and U0126 (MEK1/2 Kinase Inhibitor) #9903 for 2 hours, followed by stimulation with hEGF #8916 for 20 minutes. The phosphorylation status of S6 Ribosomal Protein, as well as the total protein expression level, was measured simultaneously using the PhosphoPlus® S6 Ribosomal Protein (Ser235/236) In-Cell Duet (ICW Compatible) #7261. With increasing concentrations of LY294002 and U0126, a significant decrease (~10-fold) in phospho-S6 signal as compared to the hEGF-stimulated control was observed, while total S6 protein levels remained unchanged and were used to normalize the data. When using phospho-S6 as a measurement, the IC50 of these compounds was 1 μM. Data and images were generated using the LI-COR® Biosciences Odyssey® Infrared Imaging System.

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Product Description

PhosphoPlus® S6 Ribosomal Protein (Ser235/236) In-Cell Duet from Cell Signaling Technology (CST) provides an easy method to assess protein activation status using a multi-well plate scanner with near infrared detection capabilities, such as the LI-COR® Biosciences Odyssey® Infrared Imaging System. This kit contains a pre-optimized activation-state and total protein antibody cocktail, selected based on superior performance. Phosphorylated and total protein are detected simultaneously in the same well, allowing levels of phosphorylated protein to be normalized to total protein. A near infrared detection cocktail is also included.


Specificity / Sensitivity

Phospho-S6 Ribosomal Protein (Ser235/236) detects endogenous levels of ribosomal protein S6 only when phosphorylated at Ser235 and 236. Total S6 Ribosomal Protein detects endogenous levels of total S6 ribosomal protein independent of phosphorylation.


Source / Purification

Monoclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser235 and Ser236 of human ribosomal protein S6 or with a GST-S6 ribosomal protein (full length) fusion protein.

One way that growth factors and mitogens effectively promote sustained cell growth and proliferation is by upregulating mRNA translation (1,2). Growth factors and mitogens induce the activation of p70 S6 kinase and the subsequent phosphorylation of the S6 ribosomal protein. Phosphorylation of S6 ribosomal protein correlates with an increase in translation of mRNA transcripts that contain an oligopyrimidine tract in their 5' untranslated regions (2). These particular mRNA transcripts (5'TOP) encode proteins involved in cell cycle progression as well as ribosomal proteins and elongation factors necessary for translation (2,3). 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 (4,5).


1.  Dufner, A. and Thomas, G. (1999) Exp Cell Res 253, 100-9.

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

3.  Jefferies, H.B. et al. (1997) EMBO J 16, 3693-704.

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

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


Entrez-Gene Id 6194
Swiss-Prot Acc. P62753


For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc.
PhosphoPlus® is a trademark of Cell Signaling Technology, Inc.
LI-COR® is a registered trademark of LI-COR, Inc.
Odyssey® is a registered trademark of LI-COR, Inc.