Cell Signaling Technology

Product Pathways - TGF-beta/Smad Signaling

Phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) (D27F4) Rabbit mAb #8828

Applications Reactivity Sensitivity MW (kDa) Isotype
W IF-IC F H M R Mk Endogenous 52, 60 Rabbit IgG

Applications Key:  W=Western Blotting  IF-IC=Immunofluorescence (Immunocytochemistry)  F=Flow Cytometry
Reactivity Key:  H=Human  M=Mouse  R=Rat  Mk=Monkey
Species cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Protocols

* Product-specific protocol.

Specificity / Sensitivity

Phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) (D27F4) Rabbit mAb recognizes endogenous levels of Smad2 protein when phosphorylated at Ser465 and Ser467. This antibody also recognizes endogenous levels of Smad3 protein when phosphorylated Ser422 only or at both Ser423 and Ser425.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ser465/467 of human Smad2 protein.

Western Blotting

Western Blotting

Western blot analysis of extracts from HaCaT cells, untreated (-) or treated with hTGF-β3 #8425 (+) in the absence or presence of the TGFR inhibitor SB 431542, using Phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) (D27F4) Rabbit mAb (upper) or Smad2/3 (D7G7) XP® Rabbit mAb #8685 (lower).

Flow Cytometry

Flow Cytometry

Flow cytometric analysis of HT-1080 cells, untreated (blue), treated with hTGF-β3 #8425 (green), or treated with hTGF-β3 and SB 431542 (red), using Phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) (D27F4) Rabbit mAb.

IF-IC

IF-IC

Confocal immunofluorescent analysis of HaCaT cells, serum-starved (left), treated with hTGF-β3 #8425 (100 ng/ml, 30 min; center), or treated with hTGF-β3 and SB 431542 (10 μg/mL, 1 hr; right), using Phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) (D27F4) Rabbit mAb (green) and COX IV (3E11) Rabbit mAb (Alexa Fluor® 555 Conjugate) #8693 (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).

  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.

Application References

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Companion Products

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

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