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Western blot analysis of extracts from serum starved or serum treated (20%) 293, NIH/3T3, and PC12 cells, using Phospho-p70 S6 Kinase (Thr389) (108D2) Rabbit mAb (upper), or p70 S6 Kinase (49D7) rabbit mAb #2708 (lower).

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Western blot analysis of extracts from 293 cells, untreated, serum-treated or EGF-treated (100 ng/ml), using Phospho-p70 S6 Kinase (Thr421/Ser424) Antibody (upper) or p70 S6 Kinase Antibody #9202 (lower).

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Western blot analysis of extracts from PC12, NIH/3T3, and SK-N-MC cells, using p70 S6 Kinase (49D7) Rabbit mAb.

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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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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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Immunoprecipitation of phosphorylated p70 S6 Kinase from 293 cell extracts (cells were serum-stimulated as indicated) under nondenaturing or denaturing conditions, followed by Western blot analysis, using Phospho-p70 S6 Kinase (Thr421/Ser424) Antibody.

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Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-), SignalSilence® p70/85 S6 Kinase siRNA I (+) or SignalSilence® p70/85 S6 Kinase siRNA II #6572 (+), using p70 S6 Kinase (49D7) Rabbit mAb #2708 and α-Tubulin (11H10) Rabbit mAb #2125. The p70 S6 Kinase (49D7) Rabbit mAb confirms silencing of p70/85 S6 kinase expression, while the α-Tubulin (11H10) Rabbit mAb is used to control for loading and specificity of p70/85 S6 kinase siRNA.

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Immunohistochemical analysis of paraffin-embedded human colon carcinoma, using p70 S6 Kinase (49D7) Rabbit mAb.

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Immunohistochemical analysis of paraffin-embedded human lung carcinoma, showing cytoplasmic and nuclear localization, using p70 S6 Kinase (49D7) Rabbit mAb.

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Immunohistochemical analysis of paraffin-embedded human breast carcinoma, using p70 S6 Kinase (49D7) Rabbit mAb in the presence of control peptide (left) or p70 S6 Kinase Blocking Peptide #1205 (right).

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Image
Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Phospho-p70 S6 Kinase (Thr389) (108D2) Rabbit mAb 9234 100 µl
Western Blotting
H M R Mk 70, 85 Rabbit IgG
Phospho-p70 S6 Kinase (Thr421/Ser424) Antibody 9204 100 µl
Western Blotting Immunoprecipitation
H M R Mk 70, 85 Rabbit 
p70 S6 Kinase (49D7) Rabbit mAb 2708 100 µl
Western Blotting Immunohistochemistry
H M R Mk 70, 85 Rabbit 
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
Western Blotting
All Goat 
Anti-biotin, HRP-linked Antibody 7075 100 µl
Goat 
Biotinylated Protein Ladder Detection Pack 7727 100 µl
 
20X LumiGLO® Reagent and 20X Peroxide 7003 5 ml each
Western Blotting
 
p70 S6 Kinase Control Cell Extracts 9203 100 µl
 

Product Description

The PhosphoPlus® p70 S6 Kinase (Thr389, Thr421/Ser424) Antibody Kit provides reagents and protocols for the rapid analysis of p70 S6 kinase phosphorylation at Thr389 and Thr421/Ser424.

p70 S6 Kinase Control Cell Extracts: Total cell extracts from MCF7 cells, prepared with or without insulin treatment to serve as positive and negative controls.


Specificity / Sensitivity

p70 S6 Kinase (49D7) Rabbit mAb #2708 detects endogenous levels of total p70 S6 Kinase. It also recognizes p85 S6 Kinase. Phospho-p70 S6 Kinase (Thr389) Antibody and Phospho-p70 S6 Kinase (Thr421/Ser424) Antibody detect endogenous levels of p70 S6 Kinase only when phosphorylated at the indicated sites. These antibodies also recognize p85 S6 Kinase when phosphorylated at the corresponding sites, Thr412 or Thr444/Ser447, respectively. Phospho-p70 S6 Kinase (Thr389) Antibody may also detect S6KII phosphorylated at Thr401.


Source / Purification

Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr421/Ser424 of human p70 S6 kinase. Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr389 of human p70 S6 Kinase, or corresponding to the sequence of human p70 S6 kinase. Antibodies are purified by Protein A and peptide affinity chromatography.

p70 S6 kinase is a mitogen activated Ser/Thr protein kinase that is required for cell growth and G1 cell cycle progression (1,2). p70 S6 kinase phosphorylates the S6 protein of the 40S ribosomal subunit and is involved in translational control of 5' oligopyrimidine tract mRNAs (1). A second isoform, p85 S6 kinase, is derived from the same gene and is identical to p70 S6 kinase except for 23 extra residues at the amino terminus, which encode a nuclear localizing signal (1). Both isoforms lie on a mitogen activated signaling pathway downstream of phosphoinositide-3 kinase (PI-3K) and the target of rapamycin, FRAP/mTOR, a pathway distinct from the Ras/MAP kinase cascade (1). The activity of p70 S6 kinase is controlled by multiple phosphorylation events located within the catalytic, linker and pseudosubstrate domains (1). Phosphorylation of Thr229 in the catalytic domain and Thr389 in the linker domain are most critical for kinase function (1). Phosphorylation of Thr389, however, most closely correlates with p70 kinase activity in vivo (3). Prior phosphorylation of Thr389 is required for the action of phosphoinositide 3-dependent protein kinase 1 (PDK1) on Thr229 (4,5). Phosphorylation of this site is stimulated by growth factors such as insulin, EGF and FGF, as well as by serum and some G-protein-coupled receptor ligands, and is blocked by wortmannin, LY294002 (PI-3K inhibitor) and rapamycin (FRAP/mTOR inhibitor) (1,6,7). Ser411, Thr421 and Ser424 lie within a Ser-Pro-rich region located in the pseudosubstrate region (1). Phosphorylation at these sites is thought to activate p70 S6 kinase via relief of pseudosubstrate suppression (1,2). Another LY294002 and rapamycin sensitive phosphorylation site, Ser371, is an in vitro substrate for mTOR and correlates well with the activity of a partially rapamycin resistant mutant p70 S6 kinase (8).


1.  Pullen, N. and Thomas, G. (1997) FEBS Lett 410, 78-82.

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

3.  Weng, Q.P. et al. (1998) J Biol Chem 273, 16621-9.

4.  Pullen, N. et al. (1998) Science 279, 707-10.

5.  Alessi, D.R. et al. (1998) Curr Biol 8, 69-81.

6.  Polakiewicz, R.D. et al. (1998) J Biol Chem 273, 23534-41.

7.  Fingar, D.C. et al. (2002) Genes Dev 16, 1472-87.

8.  Saitoh, M. et al. (2002) J Biol Chem 277, 20104-12.


Entrez-Gene Id 6198
Swiss-Prot Acc. P23443


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.
LumiGLO® is a registered trademark of Kirkegaard & Perry Laboratories.
U.S. Patent No. 5,675,063.