HTRF Human/Mouse Phospho-SMAD4 Thr277 Detection Kit HTRF®

The Phospho-SMAD4 (Thr277) assay measures SMAD4 when phosphorylated at Thr277. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer.

The Phospho-SMAD4 (Thr277) assay uses 2 labeled antibodies: one with a donor fluorophore, the other with an acceptor. The first antibody was 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 involvingthe two 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 into a 384-well low volume detection plate before the additon of the Phospho-SMAD4 (Thr277) HTRF detection reagents.

This protocol enables the cells' viability and confluence to be monitored.

Detection of Phosphorylated SMAD4 (Thr277) 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 of these different cell lines were plated in 96-well  culture plates. After a 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 the pure samples diluted ½ were transferred into a 384-well small volume plate, then 4µL of Phospho T277 SMAD4 HTRF detection reagents were added. Signals were recorded overnight.

This data enables  better cell density selection for the different cell lines. The HTRF Phospho SMAD4 (Thr277) assay is able to detect human as well as mouse phosphorylated SMAD4.

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 at 37°C.

HAPI SMAD4 KO cells (Horizon Discovery)were plated at 25,000 cells per well in a 96-well plate 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 Phospho T277 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 total SMAD4, leading to a 61% Phosphorylation decrease compared to the cells transfected with the non-targeting siRNA. Moreover, no HTRF 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 Phospho T277-SMAD4 HTRF 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 phospho SMAD4 (Thr277) assay is at least 4-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 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.