The Interaction Between CLIC3and Sedlin Protein

Objective Immunofluorescence, immunoprecipitation and GST pulldown techniques were used to study CLIC3protein and Sedlin protein interaction:CLIC3eukaryotic expression vector with FLAG-tag and eukaryotic expression vector tagged with GFP were transfected into COS7cells, and their distribution were observed. GST pulldown was used to detect CLIC3interaction with Sedlin protein in vitro. Immunoprecipitation technique was used to detect interactions between CLIC3and Sedlin protein in mammalian cells. CLIC3eukaryotic expression vector with HA tag was constructed to study the interaction of CLIC3and Sedlin by GST pulldown.Methods The plasmid containing the full length cDNA sequence of human CLIC3was used as template and construct eukaryotic expression plasmids: pcDNA3.1-CLIC3-FLAG, pCDGFP-CLIC3, pCDGFP-Sedlin, pcDNA3.1-FLAG-Sedlin. By polymerase chain reaction (PCR) amplification, restriction enzyme digestion and sequencing, the correct recombinant plasmids were transformed, purified and then transfected into COS7cells and HEK-293T cells. By immunofluorescence, we observed CLIC3expression and localization in COS7cells and CLIC3and Sedlin colocalization situation in COS7cells. pGEX-5X-3-CLIC3and pGEX-5X-3-Sedlin were constructed and the direct interaction of CLIC3and Sedlin in vitro was studied by GST pulldown assay. In order to verify the interaction of CLIC3and Sedlin in mammalian cells, the co-IP was used by co-transfection of pcDNA3.1-CLIC3-FLAG and pCDGFP-Sedlin, or pcDNA3.1-FLAG-Sedlin and pCDGFP-CLIC3into HEK-293T cells.Results Restriction Enzyme digestion and sequencing results suggested that the recombinant plasmids were successful constructed. The immunofluorescence results showed that exogenous CLIC3was expressed both in the cytoplasm and the nucleus in COS7cells, Sedlin was also expressed in the cytoplasm and the nucleus. Colocalization of CLIC3and Sedlin was found both in the cytoplasm and nucleus, though mainly in the cytoplasm. GST pulldown result showed that CLIC3interact with Sedlin in vitro. Immunoprecipitation results showed that CLIC3interact with Sedlin in HEK-293T cells.Conclusion By immunofluorescence, immunoprecipitation and GST pulldown techniques, we can conclude that the CLIC3can interact with Sedlin protein in vitro and in mammalian cells. Immunofluorescence and GST pulldown techniques were used to study CLIC4protein and Sedlin protein interaction:CLIC4eukaryotic expression vector with FLAG-tag and eukaryotic expression vector tagged with GFP were transfected into COS7cells, and their distribution were observed. GST pulldown was used to detect CLIC4interaction with Sedlin protein in vitro. CLIC4eukaryotic expression vector with HA tag was constructed to study the interaction of CLIC4and Sedlin by GST pulldown.The plasmid containing the full length cDNA sequence of human CLIC4was used as template and construct eukaryotic expression plasmids: pcDNA3.1-CLIC4-FLAG, pCDGFP-CLIC4, pCDGFP-Sedlin, pcDNA3.1-FLAG-Sedlin. By polymerase chain reaction (PCR) amplification, restriction enzyme digestion and sequencing, the correct recombinant plasmids were transformed, purified and then transfected into COS7cells and HEK-293T cells. By immunofluorescence, we observed CLIC4expression and localization in COS7cells and CLIC4and Sedlin colocalization situation in COS7cells. pGEX-5X-3-CLIC4and pGEX-5X-3-Sedlin were constructed and the direct interaction of CLIC4and Sedlin in vitro was studied by GST pulldown assay.Restriction Enzyme digestion and sequencing results suggested that the recombinant plasmids were successful constructed. The immunofluorescence results showed that exogenous CLIC4was expressed both in the cytoplasm and the nucleus in COS7cells, Sedlin was also expressed in the cytoplasm and the nucleus. Colocalization of CLIC4and Sedlin was found both in the cytoplasm and nucleus, though mainly in the cytoplasm. GST pulldown result showed that CLIC4interact with Sedlin in vitro. Conclusion By immunofluorescence and GST pulldown techniques, we can conclude that the CLIC4can interact with Sedlin protein in vitro and in mammalian cells.