Alpha-SMA quantification in cell lysates
The Total-SMAD4 detection kit detects cellular SMAD4, and can be used as a normalization assay with our Phospho-SMAD4 kit to enable optimal investigation of TGF-ß signaling.
The Total-SMAD4 cellular assay kit is designed to monitor the expression level of SMAD4, whether phosphorylated or unphosphorylated.
It is compatible with our Phospho-SMAD4 kit, and enables the analysis of phosphorylated and total proteins from a single sample for a better readout of TGF-ß signaling activity.
The Total-SMAD4 assay quantifies the expression level of SMAD4 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer.
The Total-SMAD4 assay uses two labeled antibodies: one coupled to a donor fluorophore, the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of SMAD4 in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the protein’s expression 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 the addition of the Total-SMAD4 HTRF detection reagents.
This protocol enables the cells' viability and confluence to be monitored.
Detection of total SMAD4 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 HAPI cell lines were selected to test human compatibility, while NIH 3T3 cells were chosen for mouse compatibility. 25,000, 50,000, and 100,000 cells from these different cell lines were plated in 96-well culture plates. After 48h incubation at 37°C, 5% CO2, the cell culture medium was removed and 50µL of lysis buffer were added to the wells. A lysis step was carried out, shaking gently for 30 minutes. 16µL of pure samples were transferred into a 384-well small volume plate, then 4µL of Total-SMAD4 HTRF detection reagents were added. Signals were recorded overnight.
This data enables better cell density selection for the different cell lines. The Total-SMAD4 HTRF assay is able to detect human as well as mouse SMAD4 protein.
HAPI wt were plated at 25,000 cells per well in a 96-well plate.
After an overnight incubation at 37°C, 5% CO2, the HAPI wt cells were treated with 25 nM of ON-TARGETplus siRNA (Horizon Discovery) targeting specifically SMAD2, SMAD3, and SMAD4, or with a non-targeting siRNA (included as a control). After an overnight incubation at 37°C, 5% CO2, the medium was changed for a complete culture medium, and then the cells were incubated for an additional 24h-incubation at 37°C.
HAPI SMAD4 KO cells (Horizon Discovery) were seeded at 25,000 cells/well and were incubated for 4 days.
After the incubation, the cells were lysed with 50 µL of supplemented lysis buffer #4 (1X), and 16 µL of lysates were transferred into a low volume white microplate before the addition of 4 µL of premixed HTRF Total SMAD4 detection antibodies. The HTRF signal was recorded after an overnight incubation at RT.
Cell treatment with SMAD4 siRNA led to a significant downregulation of the Total SMAD4 signal, with a 50% signal decrease compared to the cells transfected with the non-targeting SiRNA. Moreover, no signal was observed in the HAPI SMAD4 KO cell lysate.
On the other hand, no decrease in signal was observed with cells treated with SMAD2 or SMAD3 SiRNA , demonstrating the specificity of the kit. The downregulation of SMAD2 & SMAD3 was checked using our HTRF Total SMAD2 & SMAD3 detection kits, showing a 60% and 66% loss of signal respectively.
HAPI cells were cultured in complete medium at 37°C, 5% CO2 in a T175 flask to 80% confluency.
The cells were lysed with 3 mL of supplemented lysis buffer #4 (1x) for 30 min at RT under gentle shaking.
Serial dilutions of the cell lysate were performed using supplemented lysis buffer #4 (1x), and 16µL of pure sample and each dilution were transferred into a 384-well small volume microplate, before the addition of 4µL of HTRF Total-SMAD4 detection reagents. Signals were recorded overnight.
Equal amounts of lysates were loaded into a gel for a side by side comparison between HTRF and Western Blot.
In these conditions, the HTRF total SMAD4 assay is at least 16-fold more sensitive than the Western Blot.
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 and SMAD3, enabling its oligomerization with Treonine 277 phosphorylated SMAD4.
This trimeric complex then translocates to the nucleus. There, it 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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