Sedlin Is Associated With EBI3 And Enhance The EBI3 Secretion

Objective In this paper,We studied the interaction between EBI3 protein and Sedlin protein was studied,and the interaction between EBI3 protein and Sedlin protein in vitro and mammalian cells was explored by GST pull-down and immunoprecipitation.The interaction between EBI3 deglycosylation and Sedlin protein in vitro was investigated by GST pull-down.On this basis,the effect of Sedlin on the secretion of EBI3 protein was studied by overexpressing EBI3 in COS7-NC and COS7-Sedlin cell and EBI3 protein was detected in culture medium by western blot.Methods GST-Sedlin fusion protein was prepared,and the plasmids containing EBI3-FLAG,EBI3(1-135)-FLAG and EBI3(125-230)-FLAG were transfected into HEK 293 T cells.The interaction between EBI3 protein and its truncated mutant EBI3(1-135),EBI3(125-230)with Sedlin protein in vitro was investigated by GST pull-down.To study the interaction between EBI3 deglycosylation and Sedlin protein in vitro through GST pull-down.The plasmids containing EBI3-FLAG and its truncated mutant EBI3(1-135)-FLAG and EBI3(125-230)-FLAG the plasmids containing Sedlin with GFP label and HA label respectively were co-transfected into HEK 293 T cell,the interaction of EBI3 and its truncated mutant EBI3(1-135)、EBI3(125-230)with Sedlin proteins was investigated by co-immunoprecipitation.The plasmids of EBI3-FLAG and Sedlin-HA were co-transfected into COS7 cells,EBI3 and and Sedlin co-immunoprecipitation in the cell culture medium to explore whether EBI3 could bind with Sedlin after being secreted to extracellular.We transfection of different amounts of EBI3-FLAG plasmid in COS7-Sedlin and COS7-NC cell to explore the effect of Sedlin on the secretion of EBI3 protein through the detection of EBI3 protein in the cell culture medium.Results The results of GST pull-down showed that EBI3 protein and its truncated EBI3(1-135)had interaction with Sedlin protein in vitro,while the truncated EBI3(125-230)had no interaction with Sedlin protein in vitro,and the deglycosylated EBI3 protein still had interaction with Sedlin protein in vitro.The results of co-immunoprecipitation showed that EBI3 protein and its truncated EBI3(1-135)and Sedlin protein had interaction in HEK 293 T cells,while the truncated EBI3(125-230)and Sedlin protein had no interaction in HEK 293 T cells.However,the result of co-immunoprecipitation of Sedlin with EBI3 protein in cell culture medium was negative.Detection of the EBI3 protein in the culture medium of COS7-NC and COS7-Sedlin that overexpress EBI3 by western blot,and the results showed that the content of EBI3 in the culture medium of COS7-Sedlin group was higher than that in the culture medium of COS7-NC group.Conclusion In this paper,We used the experimental methods such as GST pull-down and protein co-immunoprecipitation to show that both EBI3 protein and Sedlin protein have interactions in vitro and intracellular conditions,and that the main binding sites of EBI3 protein and Sedlin protein are at the n-terminal,while the deglycosylation of EBI3 protein will not effect the interaction between EBI3 and Sedlin protein.We detect the EBI3 protein in the culture medium of COS7-NC and COS7-Sedlin overexpress EBI3 by western blot,the results indicated that Sedlin can promote the secretion of EBI3 protein.