Cell Signaling Technology

Product Pathways - TGF-beta/Smad Signaling

Phospho-Smad1 (Ser463/465)/ Smad5 (Ser463/465)/ Smad8 (Ser426/428) Antibody #9511

Applications Reactivity Sensitivity MW (kDa) Source
W IF-IC F H M R Mi X Endogenous 60 Rabbit

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

Specificity / Sensitivity

Phospho-Smad1 (Ser463/465)/Smad5 (Ser463/465)/Smad8 (Ser426/428) Antibody detects endogenous levels of Smad1 only when dually phosphorylated at serine 463 and serine 465, as well as Smad5 and Smad8 only when phosphorylated at the equivalent sites. The antibody does not cross-react with other Smad-related proteins.

Source / Purification

Polyclonal antibodies are produced by immunizing rabbits with a synthetic phospho-peptide (KLH-coupled) corresponding to residues surrounding Ser463/465 of human Smad5. Antibodies are purified by protein A and peptide affinity chromatography.

Western Blotting

Western Blotting

Western blot analysis of extracts from untransfected Xenopus cells and BMPRI/Smad1-transfected 293 cells, untreated or BMP-4-treated, using Phospho-Smad1 (Ser463/465)/Smad5 (Ser463/465)/Smad8 (Ser426/428) Antibody. (Xenopus cells provided by Dr. Malcolm Whitman, Harvard University, Massachusetts.)

Flow Cytometry

Flow Cytometry

Flow cytometric analysis of HeLa cells, untreated (blue) or BMP-treated (green), using Phospho-Smad1 (Ser463/465)/Smad5 (Ser463/465)/Smad8 (Ser426/428) Antibody compared with a nonspecific negative control antibody (red).

IF-IC

IF-IC

Confocal immunofluorescent images of HeLa cells, untreated (left) or BMP-2-treated (right), labeled with Phospho-Smad1 (Ser463/465)/ Smad5 (Ser463/465)/ Smad8 (Ser426/428) Antibody (green) and Keratin 18 (DC10) Mouse mAb #4548 (blue). Actin filaments have been labeled with Alexa Fluor® 555 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).

  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.

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This product is for in vitro research use only and is not intended for use in humans or animals. This product is not intended for use as therapeutic or in diagnostic procedures.

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