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Phospho-SMAD3 (Ser423/425) Cellular Assay Kit   HTRF® cell-based assay for quantifying phosphorylated SMAD3 in TGF-β signaling Cisbio's cell-based homogeneous HTRF® phospho-SMAD3 (Ser423/425) immunoassay is designed for exploring TGF-β pathways directly in the cell. Phospho-SMAD3 assay enables a pharmacological readout of the TGF-β pathways by the quantitative detection of SMAD3 phosphorylated on Serine 423/425. Transforming growth factor β1 (TGF-β1) is known to be both anti-inflammatory and profibrotic. Cross-talk between TGF-β/SMAD and Wnt/β-catenin pathways in epithelial-mesenchymal transition is known to take place. Furthermore, TGF-β1 induces rapid nuclear translocation of β-catenin in adult mesenchymal stem cells, which contributes to the regeneration of multiple types of mesenchymal tissues in a SMAD3-dependent manner. Overactivation of SMAD3 is involved in a number of cancers, such as colorectal, pancreatic and chronic myeloid leukemia, and cellular processes like cell cycle/growth and endocytosis. This makes phospho-SMAD3 an important readout, targeting TGF-β receptor in cancer research or virology. With its simple add-and-read protocol, the homogeneity of the HTRF format - with no wash steps - facilitates miniaturization and is ideal for high throughput screening of small molecule compounds or biotherapeutics in cells.   Features Cell-based sandwich immunoassay for TGF-β pathway readout Truly homogeneous protocol with no wash steps More convenient and faster than ELISA or WB Excellent assay robustness  and high assay performance  Low sample volume required and simple protocol   Applications Cellular detection and quantification of phosphorylated SMAD3 (Ser423/425) Pharmacology and exploration of TGF-β signaling Functional screening of TGF-β ligands Self-renewal and differentiation of mesenchymal stem cells Oncology, inflammation, virology, metabolism    Assay Principle  HTRF® - the homogeneous cell-based sandwich immunoassay   Cisbio's phospho-SMAD3 assay is based on a TR-FRET sandwich immunoassay format comprising two specific anti-SMAD3 antibodies, one labeled with a cryptate as donor and the other with d2 as acceptor. The phospho-SMAD3 antibodies bind the phosphorylated residue, and the proximity of donor and acceptor will then lead to a fluorescent TR-FRET signal. The signal intensity is proportional to the substrate phosphorylation. The protocol is optimized for a 384-well plate format, 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 phospho-SMAD3 assay kit can be run with frozen cell lysates or fresh cells in culture. After cell lysis, phospho-SMAD3 can be quantitatively detected using the HTRF phospho-SMAD3 kit reagents and most TR-FRET multimode plate readers.    Two-plate assay protocol   For added flexibility, the assay can be run under a two-plate assay protocol, where cells are plated and treated in a 96-well culture plate. For detection of phosphorylated SMAD3, lysates are subsequently transferred to a 384 small volume assay plate where the HTRF reagents are added. This also enables the monitoring of cell viability and confluence in an appropriate cell culture plate.       Product performance 1. Validation of the HTRF phospho SMAD3 cellular assay on mouse and human cell lines   Human cervical cancer HeLa cells and mouse myoblast C2C12 cells were seeded in a T175 flask in complete culture medium and incubated for 2 days at 37°C, 5% CO2 until reaching 80% confluency.   After treatment for 30 min with  5 ng/mL TGF-β1, cells were lysed with 3 mL of supplemented lysis buffer #4 and incubated for 30 min at RT under gentle shaking. Supernatants were collected after a 10 minutes centrifugation. Serial dilutions of the cell lysate were performed in the supplemented lysis buffer and 16 µL of each dilution were transferred into a 384-well low volume white microplate before adding 4 µL of the HTRF phospho-SMAD3 Ser423/425 detection reagents. HTRF signal was recorded after 4 hours incubation time. 2. TGF-β dose-response measured by the HTRF phospho-SMAD3 cellular assay 25,000 mouse myoblast C2C12 cells were plated in a 96-well plate in complete culture medium, and incubated for 24h, at 37°C, 5% CO2. Increasing concentrations of TGF-b were added and incubated for 30 min at 37°C. After cell culture medium removal, cells were lysed with 50 µL of lysis buffer for 30 min at RT under gentle shaking. 16 µL of lysate were transferred into a 384-well sv white microplate and 4 µL of the HTRF phospho-SMAD3 detection reagents were added. The HTRF signal was recorded after 4 hours incubation. Stimulation with increasing concentrations of TGF-b induces an increase in SMAD3 phosphorylation.   3. HTRF assay compared to Western Blot using phospho SMAD3 cellular assay Mouse myoblast cells C2C12 cells were grown in a T175 flask at 37°C, 5% CO2 for 48 h. After a stimulation step with 10 ng/mL TGF-β1 for 30 min, cells were lysed with 3 mL of supplemented lysis buffer for 30 min at room temperature. The soluble fractions was were then collected after a 10 min centrifugation.   Serial dilutions of the cell lysate were performed in the supplemented lysis buffer, then 16 µL of neat or diluted fractions were dispensed and analyzed side-by-side by Western Blot and by HTRF. The HTRF phospho SMAD3 assay is at least 16-fold more sensitive than the Western Blot. Using HTRF, 1,000 cells are sufficient for minimal signal detection while 16,000 cells are needed for a Western Blot signal.         Simplified pathway TGF-β (= Transforming Growth Factor-β) is the prototypic member of the TGF-β superfamily of secreted growth factors which signal through receptor serine/threonine kinases.   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.   Cellular responses triggered by TGF-β are often cell type specific.   The TGF-β/SMAD signaling pathway regulates a variety of divergent processes during early embryogenesis and adult tissue homeostasis: cell growth, apoptosis, migration, differentiation, extracellular matrix remodeling. It also plays a role in immune and inflammatory responses.   The BMP/SMAD signaling pathway plays a key role in embryogenesis, development, adult tissue homeostasis and immune responses.   Inhibitory SMAD6 and SMAD7 are involved in feedback inhibition of the pathway.   Perturbations of the TGF-β/SMAD3 signaling pathway are linked to: Cancer (e.g. colorectal, pancreatic, breast, lung, renal, melanoma) Fibrotic diseases (e.g. pulmonary fibrosis, systemic sclerosis and scleroderma) Cardiovascular diseases (e.g. Loeys-Dietz syndrome and type II Marfan syndrome)    

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