Phospho-SMAD3 (Ser423/425) & Total SMAD3 Cellular Assay Kits

Phospho- and total SMAD3 assays as readouts of TGF-β signaling activity

These cell-based assays are designed to measure total and phosphorylated SMAD3 (Ser423/425) as pharmacological readouts of TGF-β signaling activity. Thanks to the lysis buffer compatibility, both assays can be carried out in parallel on the same lysate to normalize SMAD3 phosphorylation level depending on its expression level. TGF-β signaling is mediated by TGF-β receptors that directly activate the transcription factor SMAD3 by phosphorylation at Ser423/425. Activated SMAD3 translocates to the nucleus to regulate the expression of genes involved in cell apoptosis, migration and differentiation, as well as in immune/inflammatory responses and extracellular matrix remodeling. Perturbations of TGF-β signaling are linked to oncology, auto-immunity, cardiovascular diseases, tissue inflammation and fibrosis. In NAFLDs, overactivation of the pathway contributes to the progression from NASH to liver fibrosis by promoting the activation of Hepatic Stellate Cells (HSCs) to myofibroblasts and massive hepatocyte cell death. TGF-β signaling has therefore emerged as an attractive therapeutic target.

   Check our lysis buffer compatibility      Check our species compatibility

Watch Webinar Trends in Fibrosis Research and Drug Discovery: Nonalcoholic Steatohepatitis (NASH)

Assay principle

HTRF® - the homogeneous cell-based sandwich immunoassay

Cisbio Bioassay’s phospho- and total SMAD3 assays are based on a TR-FRET sandwich immunoassay format comprising two specific anti-SMAD3 antibodies, one labeled with Eu3+cryptate (donor) and the other with d2 (acceptor). The antibodies specifically bind to SMAD3, and the proximity of donor and acceptor then leads to a fluorescent TR-FRET signal. The protocol is optimized for a 96- or 384-well low volume microplate format (20 µL final), but can easily be further miniaturized or upscaled. Only low sample volumes are needed. The detection reagents may be pre-mixed and added in a single dispensing step for direct detection. No washing is needed at any step.

The assays can be run with frozen cell lysates or fresh cells in culture. After cell lysis, endogenous phospho- or total-SMAD3 can be quantitatively detected using the HTRF phospho-SMAD3 (Ser423/425) and total SMAD3 cellular assay kit reagents and most TR-FRET multimode plate readers.

A simpler, more flexible assay protocol - adapted to your applications

Two-plate assay protocol

For added flexibility, the assay can be run under a two-plate assay protocol, where cells are plated, treated and lysed in a culture plate. For detection, lysates are subsequently transferred into a 96- or 384-well low volume detection plate where the HTRF® reagents are added. This also enables monitoring of the cells' viability and confluence in an appropriate cell culture plate.

What to expect at the bench

This video covers the principles of phospho-HTRF assays and gives guidelines for performing them, using our phospho-ERK assay as an example.

Product performances

1. Overactivation of TGF-β/SMAD signaling in hepatocytes and HSCs promotes liver fibrosis

The human hepatic stellate cell line LX-2* and the human hepatoma cell line HepG2 were plated in culture-treated 96-well plates (50,000 and 200,000 cells/well respectively) in complete culture medium and incubated overnight at 37°C - 5% CO2. The next day, the cells were treated with increasing concentrations of TGF-β1 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 human hepatic cell lines (HepG2 and LX-2*), TGF-β1 treatment induces SMAD3 activation by phosphorylation on Ser423/425, while the expression level of the protein is not significantly affected. Overactivation of TGF-β signaling promotes hepatocyte death and HSC activation into myofibroblasts, which are two cellular processes contributing to the progression from NASH to liver fibrosis.

*LX-2 cell line provided by EMD Millipore (Part #SCC064)

2. Validation on the mouse cell lines NIH/3T3 and C2C12

The mouse fibroblast cell line NIH/3T3 and the mouse myoblast cell line C2C12 were plated in culture-treated 96-well plates (100,000 cells/well) in complete culture medium and incubated at 37°C - 5% CO2. The day after, the cells were treated with increasing concentrations of TGF-β1 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 mouse cell lines, TGF-β1 promotes the activation of SMAD3 by phosphorylation on Ser423/425, whereas the expression level of the protein remains fairly stable.

3. HTRF phospho- and total SMAD3 assays compared to western-blot

Cells were seeded in T175 flasks in complete culture medium, and incubated for 2 days at 37°C, 5% CO2 until 80% confluency was reached. After treatment (or not) with 10 ng/mL TGF-β1 for 30 minutes, the cells were lysed with 3 mL of supplemented lysis buffer #4 for 30 minutes at RT under gentle shaking. Soluble supernatants were collected after a 10-minute centrifugation.

Serial dilutions of the cell lysates were performed in the supplemented lysis buffer and 16 µL of each dilution were transferred into a low volume white microplate before the addition of 4 µL of the HTRF® phospho- or total SMAD3 detection reagents. Equal amounts of lysates were used for a side by side comparison between HTRF and Western Blot.

Using HTRF® Phospho-SMAD3 detection reagents, only 1,000 cells were sufficient for minimal signal detection, while 16,000 cells were needed for a WesternBlot signal. The HTRF® phospho-SMAD3 assay is 16-fold more sensitive than the Western Blot. Using either HTRF® Total SMAD3 detection reagents or Western Blot, 1,600 cells were sufficient for minimal signal detection, demonstrating that the HTRF® total SMAD3 assay is as sensitive as the Western Blot.

Simplified pathway

TGF-β signaling is mediated by complexes of TGF-β type I and type II receptors (TβRI is also called ALK5, and TβRII) which directly activate the intracellular signaling protein SMAD3 by phosphorylation at two C-terminal serine residues (Ser423/425).

The binding of the bioactive dimeric TGF-β ligand on a homodimer of TβRII triggers the recruitment of a homodimer of TβRI into the ligand-receptor complex. TβRII, which is a constitutively active kinase, undergoes autophosphorylation and transphosphorylates TβRI. Activated TβRI in turn phosphorylates SMAD3 at Ser423 and Ser425, enabling its oligomerization with the co-mediator (Co-SMAD) SMAD4. This complex then translocates to the nucleus, where it accumulates and functions as a transcription factor with coactivators and corepressors to regulate the expression of multiple target 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.

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
SMAD3 phospho-S423/425 kit - 500 tests63ADK025PEG
SMAD3 phospho-S423/425 kit - 10,000 tests63ADK025PEH
SMAD3 phospho-S423/425 kit - 50,000 tests63ADK025PEY
-
SMAD3 total kit - 500 tests64ND3PEG
-
SMAD3 total kit - 10,000 tests64ND3PEH
-
SMAD3 total kit - 50,000 tests64ND3PEY
-

Companion products

DescriptionCat. noProduct insertMSDS
SMAD3 phospho-S423/425 kit - control lysate63ADK025TDA
-
SMAD3 total kit control lysate64ND3TDA
-

Documents