Alpha-SMA quantification in cell lysates
This HTRF cell-based assay enables rapid, quantitative detection of SMAD1 phosphorylated at Serine 463/465. After phosphorylation and translocation to the nucleus, SMAD1 acts as a transcription factor in regulating the expression of genes involved in stem cell renewal, cell proliferation, apoptosis, migration, differentiation, and immune responses.
Mouse C2C12 cells were cultured to 80% confluency. After BMP-4 treatment, cells were lysed and soluble supernatants were collected via centrifugation. Serial dilutions of the cell lysate were performed and 16 µL of each dilution were transferred into a 384-well low volume white microplate before finally adding phospho kit reagents. A side by side comparison showed the HTRF Phospho assay is at least 8-fold more sensitive than the Western Blot, and shows optimal correlation.
BMP/SMAD signaling pathway plays a key role in embryogenesis, development, adult tissue homeostasis and immune responses. It is mediated by heterotetrameric complexes of BMPRI and BMPRI, that activate the intracellular signaling proteins SMAD1/5/8 by phosphorylating two C-terminal serine residues.
BMP-2/4 binding on BMPRI triggers the recruitment of BMPRII, which then transphosphorylates the intracellular domain of BMPRI.
BMPRI in turn phosphorylates SMAD1 at Ser463 and Ser465, enabling its oligomerization with the co-mediator (Co-SMAD) SMAD4. This complex translocates to the nucleus, where it functions as a transcription factor with coactivators and corepressors to regulate gene expression.
Inhibitory SMAD6 and SMAD7 are involved in feedback inhibition of the signaling pathway.
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