西南医科大学学报2025，Vol.48Issue(1)：36-40,5.DOI:10.3969/j.issn.2096-3351.2025.01.008
基于TurboID邻近蛋白标记技术鉴定细胞内Smad3相互作用网络
Identification of Intracellular Smad3 Interaction Networks via TurboID Proximity Labeling Technology
摘要
Abstract
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.关键词
邻近标记/高效蛋白质鉴定技术/母细胞分化蛋白3/肾小管上皮细胞Key words
Proximity Labeling/Turbo identification of interacting proteins(TurboID)/Mothers against decapentaplegic homo-log 3(Smad3)/Renal tubular epithelial cells分类
基础医学引用本文复制引用
张璐娜,倪玉芳,鲜茜雯,王洪连,粟宏伟,王丽,李健春..基于TurboID邻近蛋白标记技术鉴定细胞内Smad3相互作用网络[J].西南医科大学学报,2025,48(1):36-40,5.基金项目
国家自然科学基金青年项目(82104665) （82104665）
四川省科技计划项目(2023NSFSC1763,2022YFS0621) （2023NSFSC1763,2022YFS0621）
国家级大学生创新创业项目(202310632067) （202310632067）
