Cell Signaling Technology

Product Pathways - Translational Control

PhosphoPlus® p70 S6 Kinase (Thr389, Thr421/Ser424) Antibody Kit #9430

Kit Includes Quantity Applications Reactivity MW (kDa) Isotype
Phospho-p70 S6 Kinase (Thr389) (108D2) Rabbit mAb #9234 100 µl W H M R Mk (C) 70, 85 Rabbit IgG
Phospho-p70 S6 Kinase (Thr421/Ser424) Antibody #9204 100 µl W IP H M R Mk 70, 85 Rabbit
p70 S6 Kinase (49D7) Rabbit mAb #2708 100 µl W IHC-P H M R Mk 70, 85 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody #7074 100 µl 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
p70 S6 Kinase Control Cell Extracts #9203 100 µl

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IHC-P=Immunohistochemistry (Paraffin)
Reactivity Key:  H=Human  M=Mouse  R=Rat  Mk=Monkey  C=Chicken
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

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.

Western Blotting

Western Blotting

Western blot analysis of extracts from HeLa, NIH/3T3, PC12 and COS-7 cells using p70 S6 Kinase Antibody #9202.

Western Blotting

Western Blotting

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 #9234.

IP

IP

Immunoprecipitation of extracts from serum-treated 293 cells under nondenaturing or denaturing conditions, using Phospho-p70 S6 Kinase (Thr421/Ser424) Antibody #9204. Western blot was then performed using the same antibody.


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.

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.

Background

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, 100-109.
  3. Weng, Q.P. et al. (1998) J. Biol. Chem. 273, 16621-16629.
  4. Pullen, N. et al. (1998) Science 279, 707-710.
  5. Alessi, D.R. et al. (1998) Curr. Biol. 8, 69-81.
  6. Polakiewicz, R.D. et al. (1998) J. Biol. Chem. 273, 23534-23541.
  7. Fingar, D.C. et al. (2002) Genes Dev. 16, 1472-1487.
  8. Saitoh, M. et al. (2002) J. Biol. Chem. 277, 20104-20112.

Application References

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For Research Use Only. Not For Use In Diagnostic Procedures.

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