Cell Signaling Technology
XP Monoclonal Antibody

Product Pathways - TGF-beta/Smad Signaling

Phospho-Smad2 (Ser465/467)/ Smad3 (Ser423/425) (D6G10) XP® Rabbit mAb #9510

Applications Reactivity Sensitivity Isotype
IF-IC F H Endogenous Rabbit IgG

Applications Key:  IF-IC=Immunofluorescence (Immunocytochemistry)  F=Flow Cytometry
Reactivity Key:  H=Human
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)/Smad3 (Ser423/425) (D6G10) XP® Rabbit mAb detects endogenous levels of phosporylated Smad2 and Smad3. Some reactivity is observed with non-phosphorylated Smad2 and 3 by western blot analysis. This antibody does not cross-react with other Smad-related proteins.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser465/467 of human Smad2. This region is highly conserved with Smad3 at Ser423/425.

Flow Cytometry

Flow Cytometry

Flow cytometric analysis of HT-1080 cells, untreated (blue) or TGF-β treated (green), using Phospho-Smad2 (Ser465/467/Smad3 (Ser423/425) (D6G10) XP® Rabbit mAb compared to a nonspecific negative control antibody (red).

IF-IC

IF-IC

Confocal immunofluorescent analysis of HeLa cells, serum-starved (left) or treated (right) with Human TGF-β3 #3706, using Phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) (D6G10) XP® Rabbit mAb (green). Actin filaments have been labeled with DY-554 phalloidin (red).

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-β, Smad2 and Smad3 become phosphorylated at their carboxyl termini (serines 465 and 467 on Smad2; serines 423 and 425 on Smad3) by the receptor kinase TGF-β R1(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. USA 94, 10669-10674.

Application References

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This product is intended for research purposes only. The product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

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