Alpha-SMA quantification in cell lysates
The phospho-SMAD3 (Ser423/425) kit enables the cell-based quantitative detection of SMAD3 phosphorylated on Ser423/425, as a readout of the TGFb pathway.
This HTRF cell-based assay enables the rapid, quantitative detection of Phosphorylated SMAD3 phosphorylated at Serine 423/425 as a readout of TGF-ß signaling activity. TGF-ß receptors directly activate SMAD3 by phosphorylation at Ser423/425 causing it to translocate to the nucleus and regulate gene expression involved in apoptosis, migration and differentiation, as well as in immune/inflammatory responses and extracellular matrix remodeling.
Mouse NIH/3T3 and C2C12 lines were plated (100,000 cells/well) and cultured 2h in complete culture medium, 37°C - 5% CO2. The day after, the cells were treated with increasing concentrations of TGF-ß1 for 30 minutes at 37°C - 5% CO2. After medium removal, the cells were lysed with 50 µL of supplemented lysis buffer #4 for 30 minutes at RT under gentle shaking, and 16 µL of lysate were transferred twice over into a low volume white microplate before adding 4 µL of the HTRF phospho-SMAD3 or total SMAD3 detection antibodies. The HTRF signal was recorded after an overnight incubation. In both lines, TGF-ß1 promotes the activation of SMAD3 by phosphorylation on Ser423/425, whereas the expression level of the protein remains fairly stable.
Human LX-2* and HepG2 lines were plated (50,000 and 200,000 cells/well respectively) and cultured overnight in complete culture medium, 37°C - 5% CO2. Cells were then treated with increasing concentrations of TGF-ß1 for 30 minutes at 37°C - 5% CO2. After medium removal, cells were lysed with 50 µL of supplemented lysis buffer #4 for 30 minutes at RT under gentle shaking, and 16 µL of lysate were transferred twice over into a low volume white microplate before adding 4 µL of the HTRF phospho-SMAD3 or total SMAD3 detection antibodies. The HTRF signal was recorded after an overnight incubation. In both lines, TGF-ß1 treatment induces SMAD3 phosphorylation on Ser423/425, while the expression level of the protein is not significantly affected.
*LX-2 cell line provided by EMD Millipore (Part #SCC064)
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