Cell Signaling Technology

Product Pathways - TGF-beta/Smad Signaling

Phospho-Smad1/5 (Ser463/465) (41D10) Rabbit mAb #9516

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

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

Protocols

Specificity / Sensitivity

Phospho-Smad1/5 (Ser463/465) (41D10) Rabbit mAb detects endogenous levels of Smad1/5 only when dually phosphorylated at Ser463 and Ser465. The antibody does not cross-react with other Smad-related proteins.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser463/465 of human Smad5.

Western Blotting

Western Blotting

Western blot analysis of extracts from untreated or BMP-4-treated HeLa or NIH/3T3 cells using Phospho-Smad1/5 (Ser463/465) (41D10) Rabbit mAb.

Flow Cytometry

Flow Cytometry

Flow cytometric analysis of HeLa cells, untreated (blue) or BMP-treated (green), using Phospho-Smad1/5 (Ser463/465) (41D10) Rabbit mAb compared to a nonspecific negative control antibody (red).

IF-IC

IF-IC

Confocal immunofluorescent analysis of HT-1080 cells, BMP-treated (left) and untreated (right), using Phospho-Smad5 (Ser463/Ser465) (41D10) Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red).


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

Rabbit Monoclonals Produced Using Epitomics® Technology, U.S. Patent No. 5,675,063.

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

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