The Interaction Between GTFIIF2and CLIC1Protein

Objective To investigate the interaction of general transcription factor GTFIIF2proteinand intracellular chloride channel1protein, plasmid pCDNA3.1-FLAG-GTFIIF2,pCDGFP-GTFIIF2and GEX-5X-3-GTFIIF2were constructed and transiently transfected ortransformed into cells. With immunofluorescence, GST pulldown, co-IP and othertechniques, we studied the expression and localization of GTFIIF2and CLIC1respectivelyand the colocalization and the interaction of the two proteins.Methods Design GTFIIF2upstream and downstream primers and amplify GTFIIF2fromthe template containing full-length cDNA sequence of GTFIIF2by polymerase chainreaction. Construct eukaryotic expression vector pCDNA3.1-FLAG-GTFIIF2and pCDGFP-GTFIIF2, and prokaryotic expression vector pGEX-5X-3-GTFIIF2, checked by doublerestriction enzyme digestion and sequencing. Transfect the purified correct pCDNA3.1-FLAG-GTFIIF2or pCDGFP-GTFIIF2into COS7cells respectively, observe the relativedistribution and expression. Then co-transfected the plasmids into COS7cells andinvestigated the co-localization of the GTFIIF2and CLIC1protein in mammalian cells.GEX-5X-3-GTFIIF2was correctly constructed and transformed into BL21competent cells,the fusion protein was prepared, and at the same time, pCDGFP-CLIC1was transfected intoHEK-293T cells, GST pulldown technique was used to detect the interaction in vitro.pcDNA3.1-FLAG-GTFIIF2and pCDGFP-CLIC1were co-transfected into HEK-293T cellsand co-immunoprecipitation method was used to detect the interaction in vivo.Results Restriction enzyme digestion and sequencing results showed that therecombinant plasmids pCDNA3.1-FLAG-GTFIIF2, pCDGFP-GTFIIF2and pGEX-5X-3-GTFIIF2were correctly constructed. The immunofluence results indicated that GTFIIF2mainly distributed in nucleus in COS7cells and CLIC1distributed both in nucleus and cytoplasm, and there was co-localization of the two proteins. pGEX-5X-3-GTFIIF2fusionprotein was successfully induced and expressed, GST pulldown results showed that theGTFIIF2interacted with CLIC1in vitro; By co-immunoprecipitation in HEK-293T cells,we concluded that GTFIIF2interacted with CLIC1in mammalian cells.Conclusion By immunofluorescence, GST pulldown and co-immunoprecipitationexperiments, we verified that the GTFIIF2protein and CLIC1protein can interact in vitroand in mammalian cells.