Cell Signaling Technology

Product Pathways - TGF-beta/Smad Signaling

Phospho-Smad3 (Ser423/425) (C25A9) Rabbit mAb #9520

Applications Reactivity MW (kDa) Source Isotype
W IP H (M) (R) (Mk) (B) (X) (Z) 52 Rabbit IgG

Applications Key:  W=Western Blotting  IP=Immunoprecipitation
Reactivity Key:  H=Human  M=Mouse  R=Rat  Mk=Monkey  B=Bovine  X=Xenopus  Z=Zebra Fish
Species enclosed in parentheses are predicted to react based on 100% sequence homology. Species cross-reactivity is determined by Western blot.

Specificity / Sensitivity

Phospho-Smad3 (Ser423/425) (C25A9) Rabbit mAb detects endogenous levels of Smad3 when phosphorylated at Ser423/425. This antibody does not cross-react reactivity with other family members.

Source / Purification

Rabbit monoclonal antibodies were prepared from spleens obtained from rabbits immunized with a synthetic phosphopeptide (KLH-coupled) corresponding to residues surrounding Ser423/425 of Smad3.

Western Blotting

Western Blotting

Western blot analysis of extracts from HT-1080 cells, untreated or treated with TGF-β1, TGFR inhibitor SB-431542 or BMP-2, using Phospho-Smad3 (Ser423/425) (C25A9) Rabbit mAb (top) or total Smad3 (C67H9) Rabbit mAb #9523 (bottom).

Background

Members of the Smad family of signal transduction molecules are components of a critical intracellular pathway that transmits TGF-β signals from the cell surface into the nucleus. Three distinct classes of Smads have been defined: the receptor-regulated Smads (R-Smads), which include Smad1, 2, 3, 5 and 8, the common-mediator Smad (co-Smad), Smad4, and the antagonistic or inhibitory Smads (I-Smads), Smad6 and 7 (1-5). Activated type I receptors associate with specific R-Smads and phosphorylate them on a conserved carboxy-terminal SSXS motif. The phosphorylated R-Smad dissociates from the receptor and forms a heteromeric complex with the co-Smad (Smad4), allowing translocation of the complex to the nucleus. Once in the nucleus, Smads can target a variety of DNA binding proteins to regulate transcriptional responses (6-8).

Following stimulation by TGF-β, Smad2 and Smad3 become phosphorylated at carboxyl terminal serine residues (Ser465 and 467 on Smad2; Ser423 and 425 on Smad3) by TGF-β Receptor I. Phosphorylated Smad 2/3 can complex with Smad4 and translocate to the nucleus to regulate gene expression (9-11).

  1. Heldin, C.H. et al. (1997) Nature 390, 465-471.
  2. Attisano, L. and Wrana, J.L. (1998) Curr. Opin. Cell Biol. 10, 188-194.
  3. Derynck, R. et al. (1998) Cell 95, 737-740.
  4. Massague, J. (1998) Annu. Rev. Biochem. 67, 753-791.
  5. Whitman, M. et al. (1998) Genes Dev. 12, 2445-2462.
  6. Wrana, J. (2000) Science 23, 1-9.
  7. Attisano, L. and Wrana, J. (2002) Science 296, 1646-1647.
  8. Moustakas, A. et al. (2001) J. Cell Sci. 114, 4359-4369.
  9. Abdollah, S. et al. (1997) J. Biol. Chem. 272, 27678-27685.
  10. Souchelnytskyi, S. et al. (1997) J. Biol. Chem. 272, 28107-28115.
  11. Liu, X. et al. (1997) Proc. Natl. Acad. Sci. USA 94, 10669-10674.

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