Alpha-SMA quantification in cell lysates
The phospho-SMAD2 (Ser465/467) kit enables the cell-based quantitative detection of SMAD2 phosphorylated on Ser465/467, as a readout of the TGFb pathway.
This HTRF cell-based assay enables the rapid, quantitative detection of SMAD2 phosphorylated at Serine 465/467, as a readout of TGF-ß signaling activity.
TGF-ß receptors directly activate SMAD2 by phosphorylation at Ser465/467, 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.
The Phospho-SMAD2 (Ser465/4267) assay measures SMAD2 when phosphorylated at Ser465/4267. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer.
The Phospho-SMAD2 (Ser465/4267) assay uses 2 labeled antibodies: one with a donor fluorophore, the other with an acceptor. The first antibody is selected for its specific binding to the phosphorylated motif on the protein, and the second for its ability to recognize the protein independently of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving both labeled antibodies and which brings the donor fluorophore into close proximity to the acceptor, thereby generating a FRET signal. Its intensity is directly proportional to the concentration of phosphorylated protein present in the sample, and provides a means of assessing the protein’s phosphorylation state under a no-wash assay format.
The 2 plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates to a 384-well low volume detection plate before adding Phospho-SMAD2 (Ser465/467) HTRF detection reagents.
This protocol enables the cells' viability and confluence to be monitored.
Detection of Phosphorylated SMAD2 (Ser465/467) with HTRF reagents can be performed in a single plate used for culturing, stimulation, and lysis. No washing steps are required.
This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.
HeLa and C2C12 cell lines were plated (100,000 cells/well) and cultured overnight in complete culture medium, 37°C - 5% CO2. The day after, medium was removed and the cells were incubated for 4h at 37°C - 5% CO2 with reduced culture medium (0.5% FBS). After incubation, cells were treated with increasing concentrations of TGF-ß1 for 30 minutes at 37°C - 5% CO2. After medium removal, the cells were then lysed with 50 µL of supplemented lysis buffer #1 for 30 minutes at RT under gentle shaking, and 16 µL of lysate were transferred twice over into a low volume white microplate before the addition of 4 µL of the HTRF phospho-SMAD2 or total SMAD2 detection antibodies.
The HTRF signal was recorded after an overnight incubation. In both lines, TGF-ß1 promoted the activation of SMAD2 by phosphorylation on Ser465/467, whereas the expression level of the protein remained fairly stable.
TGF-ß signaling is mediated by complexes of TßRI and TßRII, which activate intracellular SMAD3 and SMAD2 by phosphorylation. The binding of the TGF-ß ligand on TßRII triggers the recruitment of TßRI into the ligand-receptor complex. TßRII autophosphorylates, then transphosphorylates TßRI. Activated TßRI in turn phosphorylates SMAD2 on Ser465 and Ser467, enabling its oligomerization with SMAD4. This complex then translocates into the nucleus, and acts as a transcription factor with coactivators and corepressors to regulate the expression of multiple genes involved in cell growth, apoptosis, proliferation, migration, and differentiation, as well as in extracellular matrix remodeling and immune/inflammatory responses. Inhibitory SMAD6 and SMAD7 are involved in feedback inhibition of the pathway.
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