The Study On The Interaction Of MRG15with Sedlin

Objective To investigate interaction between the cellular senescence protein (MRG15）and the spondyloepiphyseal dysplasia tarda protein Sedlin. We used yeast two hybridsystem， GST pull-down technique， indirect immunofluorescence technique，co-immunoprecipitation technique to investigate the interaction between MRG15andSedlin.Methods We used yeast two hybrid system to test interaction between MRG15andSedlin.The full length cDNA of MRG15was amplified with PCR,to conduct into theshuttle vector of pGADT7, then forming pGADT7-MRG15. Then co-transformationpGADT7-MRG15and pGBKT7-Sedlin into AH109competent cells. Transformantswere screened on SD(-Leu/-Trp/-His/3AT/X-α-gal) plate.GST pull-down was to define the interaction between MRG15and Sedlin in vitro.Indirect immunofluorescence microscopy was conducted to observe localization ofpcDNA3.1-FLAG-MRG15or pcDGFP-Sedlin and colocalization between MRG15and Sedlin.The interaction of MRG15and Sedlin in mammalian cells was performed by CoIP method. The plasmids pcDGFP-Sedlin and pcDNA3.1-FLAG-MRG15wastransfected into HEK293T cells, then, the lysate was incubated with FLAG M2monoclonal antibody. and subjected to western blotting..Results Yeast two hybrid assay demonstrate MRG15interacts.with Sedlin.Immunofluorescence microscopy observed that MRG15located in the nuclei, Sedlinis located in the nucleus and cytoplasm, and these proteins were co-located in thenuclei. The interaction of MRG-15with Sedlin in vitro and in vivo was confirmed byGST pull-down and CoIP experiments, respectively.Conclusion The results showed that MRG15protein and Sedlin protein can interactwith each other,suggesting Sedlin may play a key role in cell nuclei.