Product Pathways - TGF-beta/Smad Signaling
Phospho-Smad1 (Ser463/465)/ Smad5 (Ser463/465)/ Smad8 (Ser426/428) Antibody #9511
|W IP ChIP||H M R Mi X||Endogenous||60||Rabbit|
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.
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 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 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.
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).
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- Hoodless, P.A. et al. (1996) Cell 85, 489-500.
- Klemm, J.D. et al. (1998) Annu. Rev. Immunol. 16, 569-592.
- Kretzschmar, M. et al. (1997) Genes Dev. 11, 984-995.
- Whitman, M. (1998) Genes Dev. 12, 2445-2462.
- Sapkota, G. et al. (2007) Mol Cell 25, 441-54.
- Alarcón, C. et al. (2009) Cell 139, 757-69.
- Seto, H. et al. (2004) Distinct roles of Smad pathways and p38 pathways in cartilage-specific gene expression in synovial fibroblasts. J. Clin. Invest. 113, 718-726. Applications: Western Blotting
- Watabe, T. et al. (2003) TGF-beta receptor kinase inhibitor enhances growth and integrity of embryonic stem cell-derived endothelial cells. J. Cell Biol. 163, 1303-1311. Applications: Western Blotting
- Kondo, M. et al. (2004) Cell Death Differ 11, 1092-101. Applications: Western Blotting
- Meirelles, K. et al. (2012) Proc Natl Acad Sci U S A 109, 2358-63. Applications: Western Blotting
- Wang, J. et al. (2013) J Biol Chem 288, 10418-26. Applications: ChIP
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For Research Use Only. Not For Use In Diagnostic Procedures.