Cell Signaling Technology
XP Monoclonal Antibody

Product Pathways - TGF-beta/Smad Signaling

Smad1 (D59D7) XP® Rabbit mAb #6944

Applications Reactivity Sensitivity MW (kDa) Isotype
W IP IF-IC F ChIP H M Mk (X) (B) Endogenous 60 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  Mk=Monkey  X=Xenopus  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

Smad1 (D59D7) XP® Rabbit mAb recognizes endogenous levels of total Smad1 protein.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ser190 of human Smad1 protein.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using Smad1 (D59D7) XP® Rabbit mAb.

Flow Cytometry

Flow Cytometry

Flow cytometric analysis of HT-1080 cells using Smad1 (D59D7) XP® Rabbit mAb (blue) compared to a nonspecific negative control antibody (red).

IF-IC

IF-IC

Confocal immunofluorescent analysis of HT-1080 cells, untreated (left) or treated with human BMP2 #4697 (right), using Smad1 (D59D7) XP® Rabbit mAb (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 MCF7 cells treated with Human BMP2 #4697 (50 ng/ml) for one hour and either 5 μl of Smad1 (D59D7) XP® 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 ID1 Promoter Primers #5139, human SMAD6 promoter 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.

Background

Bone morphogenetic proteins (BMPs) constitute a large family of signaling molecules that regulate a wide range of critical processes including morphogenesis, cell-fate determination, proliferation, differentiation, and apoptosis (1,2). BMP receptors are members of the TGF-β family of Ser/Thr kinase receptors. Ligand binding induces multimerization, autophosphorylation, and activation of these receptors (3-5). They subsequently phosphorylate Smad1 at Ser463 and Ser465 in the carboxy-terminal motif SSXS, as well as Smad5 and Smad8 at their corresponding sites. These phosphorylated Smads dimerize with the coactivating Smad4 and translocate to the nucleus, where they stimulate transcription of target genes (5).MAP kinases and CDKs 8 and 9 phosphorylate residues in the linker region of Smad1, including Ser206. The phosphorylation of Ser206 recruits Smurf1 to the linker region and leads to the degradation of Smad1 (6). Phosphorylation of this site also promotes Smad1 transcriptional action by recruiting YAP to the linker region (7).

  1. Hogan, B.L. et al. (1996) Genes Dev. 10, 1580-1594.
  2. Hoodless, P.A. et al. (1996) Cell 85, 489-500.
  3. Klemm, J.D. et al. (1998) Annu. Rev. Immunol. 16, 569-592.
  4. Kretzschmar, M. et al. (1997) Genes Dev. 11, 984-995.
  5. Whitman, M. (1998) Genes Dev. 12, 2445-2462.
  6. Sapkota, G. et al. (2007) Mol Cell 25, 441-54.
  7. Alarcón, C. et al. (2009) Cell 139, 757-69.

Application References

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Companion Products

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

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