| Objective To investigate the co-localization and interaction with RACK1 mutant proteins and chloride ion channel protein(CLIC1), using molecular cloning techniques, the receptor for activated C kinase 1(RACK1) mutant constructed into the eukaryotic expression plasmid, study the expression and cellular localization of RACK1 mutants and their co-localization with chloride intracellular channel protein 1(CLIC1) into eukaryocyte.Methods In pc DNA3.1-RACK1-FLAG as the template, construction of eukaryotic expression plasmid pc DNA3.1-RACK1(51-317)-FLAG, pc DNA3.1- RACK1(93-317)-FLAG, pc DNA3.1-RACK1(135-317)-FLAG, pc DNA3.1-RACK1(180-317)- FLAG, pc DNA3.1- RACK1(219-317) –FLAG, agarose gel electrophoresis to detect recombinant plasmids whether to build success. The cellular expression RACK1 mutants in HEK 293 T cells were detected by Western blotting and the localization of RACK1 mutants in COS7 cells were detected by confocal fluorescence microscopy respectively. Immunoprecipitation method further explore RACK1 deletion mutant protein and CLIC1 protein interactions, the interaction of the endogenous protein RACK1 CLIC1 protein.Results The eukaryotic expression plasmid of RACK1 mutants was successfully constructed. Western blot analysis detected remaining mutants were expressed in HEK 293 T cells lysates except pc DNA3.1-RACK1(219-317)-FLAG. Remaining mutants pc DNA3.1- RACK1(51-317)- FLAG, pc DNA3.1-RACK1(93-317)-FLAG, pc DNA3.1- RACK1(135-317)- FLAG, pc DNA3.1- RACK1(180-317)- FLAG, pc DNA3.1-RACK1(219-317)-FLAG were expressed in HEK 293 T cells precipitation. Immunofluorescence experiments show, RACK1 mutants mainly localized in the cytoplasm in COS 7 cells, the nucleus also has a small position, the co-localization existed between RACK1 mutants and CLIC1. Co-immunoprecipitation experiments demonstrated that endogenous protein CLIC1 protein interacted RACK1, deletion mutants of pc DNA3.1-RACK1(51-317)-FLAG, pc DNA3.1-RACK1(93-317)-FLAG, pc DNA3.1-RACK1(135-317)-FLAG, pc DNA3.1-RACK1(180- 317)-FLAG CLIC1 protein and protein interactions exist.Conclusion The eukaryotic expression plasmid of RACK1 mutant was successfully constructed and successfully expressed in 293 T HEK cells. All of the RACK1 mutants and CLIC1 protein had partial co-localization in COS7 cells, suggesting a possible interaction between the two proteins. It laid the foundation for the function of the protein RACK1. Co-immunoprecipitation experiments further show that the endogenous protein and RACK1 deletion mutant protein and CLIC1 protein interactions in the presence of the body. |
