Technical Data
Smad1, phosphorylated (Ser463,465) (Small Mothers Against Decapentaplegic Deleted in Pancreatic Carcinoma)
The Drosophila gene Mothers against dpp (Mad) and the C. elegans sma genes are implicated in the signaling pathway downstream of Ser/Thr kinase receptors required for signaling by the TGFb superfamily. Phosphorylation of Smad1, a human homolog of MAD is regulated and rapidly induced by bone morphogenic proteins (BMP) but not TGFb or activin. BMPs play a role in ectopic bone formation and inductive interactions during the early development of both vertebrates and invertebrates. In the mouse, deletion of the BMP4 gene leads to mesoderm formation and early embryonic lethality. Phosphorylation of MADR1 in a BMP dependent manner is functionally important in that it results in accumulation of Smad1 in the nucleus, indicative of Smad and Smad-related molecules as essential components of the Ser/Thr kinase receptor signaling pathway.

Suitable for use in Western Blot. Other applications not tested.

Recommended Dilution:
Western Blot: 0.5-2ug/ml detected phosphorylated Smad1 in RIPA lysates from P19 (mouse embryonal carcinoma) cells treated with BMP4. P19 cell lysate treated with BMP4 was resolved by electrophoresis, transferred to nitrocellulose and probed with phospho-Smad1 (Ser463/465) (1ug/ml). Proteins were visualized using a goat anti-rabbit secondary antibody conjugated to HRP and a chemiluminescence detection system.
Note: A non-specific band was detected in P19 cell lysate, Mr 34-35kD.
Optimal dilutions to be determined by the researcher.

Positive Control:
BMP-4-treated HeLa or NIH/3T3 cells

Storage and Stability:
May be stored at 4°C for short-term only. Aliquot to avoid repeated freezing and thawing. Store at -20°C. Aliquots are stable for 12 months. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
PabIgGAffinity Purified
100ug-20°CBlue IceHumanRabbit
Synthetic peptide (CG-GSPHNPIS[pS]V[pS]-GG) corresponding to aa455-465 of human Smad1 protein, conjugated to KLH.
Purified by Protein A affinity chromatography.
Supplied as a liquid in 0.1M Tris-glycine, pH 7.2, 0.15M sodium chloride, 0.05% sodium azide, 30% glycerol.
Recognizes the dual serine phosphorylated human Smad1 (Ser 463/465), which in the phosphorylated state has a MW of ~60-65kD. Species Crossreactivity: Mouse. Predicted cross-reactivity with human, rat and Xenopus.
Intended for research use only. Not for use in human, therapeutic, or diagnostic applications.
1. Macías-Silva, M., et al., J. Biol. Chem. 40: 25,628–25,636 (1998). 2. Wrana J., and Pawson T., Nature 388: 28–29 (1997). 3. Hoodless, P.A., et al., Cell 85: 489–500 (1996). 4. Macías-Silva, M., et al., Cell 87: 1215–1224 (1996).