Cell Signaling Technology

Product Pathways - TGF-beta/Smad Signaling

Phospho-Smad2 (Ser465/467) Antibody #3101

Applications Reactivity Sensitivity MW (kDa) Source
W H M R (C) (X) (Z) Endogenous 60 Rabbit

Applications Key:  W=Western Blotting
Reactivity Key:  H=Human  M=Mouse  R=Rat  C=Chicken  X=Xenopus  Z=Zebrafish
Species cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Protocols

Specificity / Sensitivity

Phospho-Smad2 (Ser465/467) Antibody detects endogenous levels of Smad2 only when dually phosphorylated at Ser465 and Ser467, and may detect phosphorylated Smad3 at its equivalent site. This antibody does not cross-react with other Smad-related proteins.

Source / Purification

Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser465/467 of human Smad2. Antibodies are purified by protein A and peptide affinity chromatography.

Western Blotting

Western Blotting

Western blot analysis of extracts from HepG2 cells treated with TGFbeta for the indicated times, using Phospho-Smad2 (Ser465/467) Antibody (upper) or Smad2 antibody (lower).

Background

Members of the Smad family of signal transduction molecules are components of a critical intracellular pathway that transmit 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-beta, Smad2 and Smad3 become phosphorylated at their carboxyl termini by the receptor kinase (serines 465 and 467 on Smad2; serines 423 and 425 on Smad3) by TbetaR-I (9-11). Following phosphorylation, Smad2 and Smad3 form a heteromeric complex with the co-smad family member Smad4. These complexes are translocated to the nucleus where they bind DNA and regulate gene transcription.

  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. (1998) Genes Dev. 12, 2445-2462.
  6. Wu, G. et al. (2000) Science 287, 92-97.
  7. Attisano, L. and Wrana, J.L. (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. 94, 10669-10674.

Application References

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Companion Products

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

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