The Interaction Protein Of Transcription Factor Smad3 Was Studied Based On TurboID Proximity Labeling Technology

Objective:To construct an interaction protein system that screens the transcription factor Smad3 by proximity labeling technology,and to verify the interaction protein function of Smad3 and its specific functions.Methods:A plasmid carrying both the sequences of the Gene of Interest Smad3 and Turbo ID was constructed,and then transfected into astrocyte U251 by lentiviral infection technique to form an overexpressed stable cell line.Based on this stable cell line,biotin labeling experiments are carried out,and after identification by silver staining,the affinity purified protein complex with biotin label is analyzed by label-free quantitative protein profiling,false positive control is eliminated,the remaining is bioinformatics analysis,the Smad3 interaction candidate protein of interest is screened,and the screened Smad3 interaction candidate protein is verified by IP and cellular immunofluorescence experiments,and passed Western The blotting experiment verifies the function of the related protein.Results: The Smad3-Turbo ID overexpression stable cell line was successfully constructed,the biotin labeling test was carried out,the Smad3 interaction candidate protein hn RNPM was screened by mass spectrometry and bioinformatics analysis,and the interaction between Smad3 and hn RNPM was verified by IP and immunofluorescence experiments,and further WB experiments were found that Smad3 had a certain role in promoting the expression of hn RNPM gene,which in turn had an inhibitory effect on IL-6.Conclusion:Due to the strong influence of smad3 gene in various fields,combined with Turbo ID,a technology with high catalytic efficiency in the field of adjacent markers,a large number of interaction proteins with the target gene Smad3 can be quickly found by using this technology.A large number of interaction proteins were found through biotin labeling experiments on Smad3-Turbo ID overexpression of stable cell lines.Follow-up experiments have verified that Smad3 does have a certain impact on hn RNPM and IL-6,laying a foundation for further study of the function of smad3 gene and the regulatory network in which it participates.