Development of an automated high‐content immunofluorescence assay of pSmads quantification: Proof‐of‐concept with drugs inhibiting the BMP/TGF‐β pathways

We performed high-content kinetic studies of pSmad expression for cells cultured in 96-well microplates in response to soluble and matrix-bound BMP-2 and TGF-β1. The detection limit of the IF-based assay was found to be similar to Western blot. Additionally, we provide a proof-of-concept for drug testing using inhibitors of BMP and TGF-β receptors, under conditions where specific signaling pathways are engaged via the ligand/receptor interactions. Altogether, our findings offer perspectives for future mechanistic studies on cell signaling and for studies at the single cell level using imaging methods.