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 IP ChIP H M R Mi X Endogenous 60 Rabbit

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  ChIP=Chromatin IP
Reactivity Key:  H=Human  M=Mouse  R=Rat  Mi=Mink  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

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 animals with a synthetic phosphopeptide 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.)

Chromatin IP

Chromatin IP

Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 MCF-7 cells treated with Human BMP2 #4697 (50 ng/ml) for 1 h and either 10 μl of Phospho-Smad1 (Ser463/465)/ Smad5 (Ser463/465)/ Smad8 (Ser426/428) 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 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.

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