Identification of Intracellular Smad3 Interaction Networks via TurboID Proximity Labeling Technology
Objective This study aimed to utilize biotin-based proximity labeling mediated by the TurboID enzyme to map the activity regulation network of the transcription factor Smad3.Methods This study constructed an inducible expression plasmid con-taining Smad3 and TurboID gene sequences,and integrated it into Smad3 knockout renal tubular epithelial cells(TCMK1)via lentivirus-mediated transfection,forming an overexpressed stable transgenic cell line.Upon stabilizing the transduction,the research team optimized the biotin labeling concentration and duration before conducting the biotin-labeling experiments.Biotinylated protein complexes were enriched and purified using streptavidin magnetic beads,followed by proteomic analysis to identify candidate proteins interacting with Smad3.Results A SSmad3-TurboID inducible overexpressed stable cell line was successfully constructed in Smad3 knockout TCMK1 cells.The optimal biotin concentration and labeling duration were established at 25 pm and 20 minutes,respectively.Proteomic analysis filtered out a series of potential Smad3 interacting candidate proteins,examples include Yap1 and Stat3.Conclu-sion Employing TurboID-mediated proximity labeling,this study successfully identified numerous candidate proteins that potentially interact with Smad3.These discoveries provided a solid foundation for future in-depth research into the functions of Smad3 protein and its regulatory network within the cell.Identification and functional analysis of these interacting proteins will lead to a more comprehen-sive understanding of the biological roles of Smad3.
Proximity LabelingTurbo identification of interacting proteins(TurboID)Mothers against decapentaplegic homo-log 3(Smad3)Renal tubular epithelial cells
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