The Study On The Interaction Between RB1and Sedlin Protein

Objective This study aims to inverstigate whether RB1protein interact with Sedlinprotein in cells. Yeast two-hybrid, GST pull-down, indirect immunofluorescence andco-immunoprecipitation.was carried out to demonstrate the interaction between RB1and Sedlin in vitro and in vivo,Methods Firstly, The full-length sequence of wild-type RB1and its deletion mutantsRB1-N、RB1-P、RB1-C were amplified, and amplified sequences inserted the pGADT7yeast expression vector or pCDNA3.1vector tagged with FLAG. In-framed constructswere confirmed by specific restriction enzymes digestion and sequencing. Secondly, thecotransformation of pGADT7-RB1and pGBKT7-sedlin into yeast AH109strain wasperformed on SD/-Leu/-Trp/-His/3AT/X-α-gal solid medium. Thirdly, plasmid ofpCDNA3.1-FLAG-RB1and each of its deletion mutants was transfected into HEK293T cells, respectively, and GST-Sedlin fusion protein was incubated with each lysatethe above,then GST pull-down assay was performed. Forthly, each plasmid of Sedlinand RB1(including its deletion mutants) tagged with FLAG or GFP was transfectedinto COS7cells to observe the localizations of the various proteins in the cells byfluorescence microscopy. The plasmids of pCDNA3.1-FLAG-RB1(including itsdeletion mutants) and pCDGFP-Sedlin were also transfected into COS7cells to observethe colocalizations of the overexpressed proteins by fluorescence microscopy. Lastly,co-immunoprecipitation assay was performed with the cotransfection ofpCDNA3.1-FLAG-RB1(including its deletion mutants) and pCDGFP-Sedlin into HEK293T cells Results Constructed plasmids the above mentioned were in correct frame byrestriction enzyme digestion and sequencing. Yeast two-hybrid assay shows RB1interacts with Sedlin in yeast cells. GST Pull-down assay shows that both RB1isassociated with RB1-C in vitro. Fruthmore, RB1protein was found mainly in the nucleiof COS7cells with indirect immunofluorescence assay, and Sedlin protein both in thenuclei and cytoplasm of COS7cells. Localizations of deletion mutants of RB1in COS7cells appears to be distributed in cytoplasm, though mainly in the nuclei. Partialcolocalization of RB1and Sedlin in the nuclei, and RB1-C and Sedlin in the nuclei. Co-immunoprecipitation assay shows that both RB1or RB1-C interacts with Sedlin in HEK293T cells.Conclusion Yeast two-hybrid、GST pull-down、indirect immunofluorescence andCo-immunoprecipitation show that the RB1protein can interacts with Sedlin protein invitro and in vivo, Sedlin is associated with C-terminal of RB1. Those suggest Sedlinmay play a critical role in cell cycle.