Cell Signaling Technology

Product Pathways - TGF-beta/Smad Signaling

Smad2/3 Antibody #5678

Applications Reactivity Sensitivity MW (kDa) Source
W IP IF-IC F ChIP H M R Mk (X) Endogenous 52, 60 Rabbit

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
Species cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Protocols

Specificity / Sensitivity

Smad2/3 Antibody recognizes endogenous levels of total Smad2/3 protein and overexpressed Smad2/3 protein.

Source / Purification

Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ala208 of human Smad2 protein. Antibodies are purified by protein A and peptide affinity chromatography.

Western Blotting

Western Blotting

Western blot analysis of extracts from ACHN and HeLa cells using Smad2/3 Antibody.

Flow Cytometry

Flow Cytometry

Flow cytometric analysis of HT-1080 cells using Smad2/3 Antibody (blue) compared to a Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (red).

IF-IC

IF-IC

Confocal immunofluorescent analysis of HT-1080 cells, serum-starved (left), treated with Human TGF-β1 #8915 (1 μg/mL for 30 min; center) or treated with Human TGF-β1 and SB43152 (10 μg/mL for 30 min; right), using Smad2/Smad3 Antibody (green). Actin filaments were labeled with DY-554 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 Smad2/3 Antibody 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).

  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.

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