Cell Signaling Technology

Product Pathways - TGF-beta/Smad Signaling

Smad3 (C67H9) Rabbit mAb #9523

Applications Reactivity Sensitivity MW (kDa) Isotype
W IP IF-IC F ChIP H M R Mk (X) (Z) (B) Endogenous 52 Rabbit IgG

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

Protocols

Specificity / Sensitivity

Smad3 (C67H9) Rabbit mAb detects endogenous levels of total Smad3 protein. No cross reactivity was detected with other family members.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues at the amino terminus of Smad3.

Western Blotting

Western Blotting

Western blot analysis of extracts from HT1080 (human), C2C12 (mouse) and B35 (rat) using Smad3 (C67H9) Rabbit mAb.

Western Blotting

Western Blotting

Western blot analysis of extracts from HT1080 cells, treated with TGF-β1, TGFR inhibitor SB-431542 or BMP-2, using Phospho-Smad3 (Ser423/425) (C25A9) Rabbit mAb #9520 (upper) or total Smad3 (C67H9) Rabbit mAb #9523 (lower).

Flow Cytometry

Flow Cytometry

Flow cytometric analysis of HT-1080 cells using Smad3 (C67H9) Rabbit mAb #9523 (blue) compared to a nonspecific negative control antibody (red).


IF-IC

IF-IC

Confocal immunofluorescent analysis of HT1080 cells, untreated (left) or TGFβ-treated (right), using Smad3 (C67H9) Rabbit mAb (green). Actin filaments have been labeled with Alexa Fluor® 555 phalloidin (red).

Chromatin IP

Chromatin IP

Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 HaCaT cells treated with Human TGF-β3 #3706 (7 ng/ml) for 1 h and either 10 μl of Smad3 (C67H9) Rabbit mAb or 2 μl of Normal Rabbit IgG #2729 using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human CDKN1A Intron 1 Primers #4669, SimpleChIP® Human ID1 Promoter Primers #5139, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.

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 (Ser465 and 467 on Smad2; Ser423 and 425 on Smad3) by TGF-β Receptor I. Phosphorylated Smad 2/3 can complex with Smad4, translocate to the nucleus and 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. (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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