Co-IP With MS For Investigating The Interacting Protein Of TRIM3

Identifying the partners of a given protein(the interactome)may provide leads about the protein's function and the molecular mechanisms in which it is involved.One of the alternative strategies used to characterize protein interactomes consists of co-immunoprecipitation(co-IP)followed by shotgun mass spectrometry(MS).This enables the isolation and identification of a protein target in its native state and its interactome from cells or tissue lysates under physiological conditions.Using co-immunoprecipitation coupled to mass spectrometry analysis,we examined the interacting protein of TRIM3.Stringent analysis of these date,and do molecular biology experiment to verify the result.The main works of this paper are follows:1.Co-IP with MS for investigating the interacting protein of TRIM3.The analysis cellular component?biological process and molecular function of the interactome protein.A comprehensive interactome of TRIM3 would serve as a powerful resourse to uncover the function of TRIM3 and provide necessary mechanistic insights.We developed 227 protein which can intereact with TRIM3,and part of protein is key member of p53 signaling pathway.2.The explored the interaction between TRIM3 and p53 by Western Blot assay.Decrease in the expression of p53 was induced by the overexpression of TRIM3 in Si Ha and ZR-75-30 cells and in a dose-dependent manner.In addition,TRIM3 could induction the transcription activity of p53 downstream target p21.3.Study on the function of TRIM3 in cervical and breast cancer.Overexpression of TRIM3 inhibited the proliferation in He La cells by MTT and colony formation assays.But overexpression of TRIM3 couldn't affect the migration of He La cells.Overexpression of TRIM3 couldn't affect the cell cycles of He La cells,but with the presence of p53,TRIM3 could affect the cell cycles of He La cells.Thus,TRIM3 could affect the cell cycles in a p53-dependent manner.Collectively,we identified the connection of TRIM3 and p53,and found TRIM3 could decreased the expression of p53.Hence,TRIM3 could inhibited the proliferation and cell cycle of He La cells.In addition,TRIM3 also inhibited the proliferation of breast cancer cells.The findings would provide a better insight for conceiving a way to combat cancer and may even provide a therapeutic strategy to contral the growth of these cancer cells.