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.