目的 利用生物素标记酶TurboID介导的邻近标记技术，筛选并构建转录因子Smad3的活性调控网络。 方法 构建一个含有Smad3和TurboID基因序列的诱导型表达质粒，并通过慢病毒介导的转染方法将其导入Smad3敲除的肾小管上皮细胞TCMK1中，形成过表达的稳定转染细胞系。在建立稳定转染细胞系的基础上，首先确定了适合的生物素标记浓度和时间，随后进行了生物素标记实验。通过链霉亲和素磁珠对带有生物素标记的蛋白复合物进行富集和纯化，随后进行蛋白质谱分析，并筛选出与Smad3相互作用的候选蛋白。 结果 在Smad3敲除的肾小管上皮细胞TCMK1中成功构建了Smad3-TurboID诱导型过表达稳转细胞株。经过生物素标记实验优化，最终确定的生物素浓度和标记时间为25 μm和20 min。质谱结果分析筛选出一系列潜在和Smad3相互作为候选蛋白，例如Yap1和Stat3。 结论 结合生物素标记酶TurboID介导的邻近标记技术，本研究成功地鉴定了大量可能与Smad3相互作用的候选蛋白。这些发现为后续深入研究Smad3蛋白的功能及其在细胞内的调控网络提供了理论基础。通过这些互作蛋白的识别和功能分析，将有助于更全面地理解Smad3的生物学作用。

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 containing 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 Smad3-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 μm and 20 minutes， respectively. Proteomic analysis filtered out a series of potential Smad3 interacting candidate proteins， examples include Yap1 and Stat3. Conclusion 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 comprehensive understanding of the biological roles ofSmad3.