Phospho-Smad3 (Ser423/425) (C2

9520:

ChIP Agarose , ChIP Magnetic , Immunoprecipitation , Western Blotting

Phospho-Smad3 (Ser423/425) (C25A9) Rabbit mAb detects endogenous levels of Smad3 when phosphorylated at Ser423/425. This antibody does not cross-react with other family members.

Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser423/425 of Smad3.

Western Blotting

Western blot analysis of extracts from HT-1080 cells, untreated or treated with TGF-β1, TGFR inhibitor SB-431542 or BMP-2, using Phospho-Smad3 (Ser423/425) (C25A9) Rabbit mAb (top) or total Smad3 (C67H9) Rabbit mAb #9523 (bottom).

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 5 μl of Phospho-Smad3 (Ser423/425) (C25A9) 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, human c-Myc intron 1 primers, 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.

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

• Louafi, F. et al. (2010) J Biol Chem 285, 41328-36. Applications: Western Blotting

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• 9523 Smad3 (C67H9) Rabbit mAb
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• 3108 Phospho-Smad2 (Ser465/467) (138D4) Rabbit mAb
• 3102 Smad2/3 Antibody
• 3122 Smad2 (86F7) Rabbit mAb
• 3103 Smad2 (L16D3) Mouse mAb
• 7071 Phototope^® -HRP Western Blot Detection System, Anti-rabbit IgG, HRP-linked Antibody
• 7074 Anti-rabbit IgG, HRP-linked Antibody
• 7720 Prestained Protein Marker, Broad Range (Premixed Format)
• 7727 Biotinylated Protein Ladder Detection Pack
• 7003 20X LumiGLO^® Reagent and 20X Peroxide

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