| Objective Construct yeast expression vectors of FKBP3for two-hybrid experiment.Construct mammalian cell expression vectors of FKBP3and CLIC1, both tagged withGFP or FLAG. Observe the colocalization of FKBP3protein and CLIC1protein. Detectwhether FKBP3protein can interact with CLIC1protein in mammalian cells.Methods pGBKT7-FKBP3was used as template to PCR amplify different fragments ofthe DNA sequences of FKBP3, recovered target fragments were inserted into pGBKT7vector to construct yeast expression vectors. Recombinant plasmid were confirmed byenzyme digestion and DNA sequencing. PCR amplified full length FKBP3and CLIC1were recovered, each fragment was inserted into either pcDNA3.1with FLAG tag orGFP vector. Constructed expression plasmids of pcDNA3.1-FKBP3-FLAG,pcDNA3.1-FLAG-CLIC1, pcDNA3.1-CLIC1-FLAG and pCD-GFP-FKBP3weretransiently transfected into COS-7cells respectively and observed by fluorescencemicroscope. FKBP3and CLIC1with different tags were co-transfected into COS-7cellsand images observed. Finally, both pcDNA3.1-FKBP3-FLAG and pCD-GFP-CLIC1were transfected into HEK293T cells, the interaction between FKBP3and CLIC1wasconfirmed by co-immunoprecipitation.Results All recombined plasmids above mentioned were constructed correctly.Immunofluorescence microscopy experiments showed FKBP3protein was distributedmainly in cytoplasma and the nuclear membrane, GFP-tagged CLIC1mainly distributedin nucleus, while FLAG tagged CLIC1, either N-terminal tag or C-terminal tag,distributed mainly in cytoplasma, less in nucleus. Immunofluorescence images of FKBP3and CLIC1in COS-7cells showed co-localization in the cytoplasma andnuclear membrane in ring-like shape. Furethermore, co-immunoprecipitation analysisshowed that FKBP3protein can interact with CLIC1protein in HEK293T cells.Conclusion FKBP3protein have a co-localization with CLIC1at cytoplasma in COS-7cells. FKBP3protein can interact with CLIC1protein in HEK293T cells. |
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