Neuritin Mediates Ubiquitination Of Ligand Jagl By Interaction With Neuralized

Object:To identify effects of Neuritin on Notch pathway mediated by Neuralized and mechanisms of Neuritin in the nervous system diseases, the protein expression levels of Neuralized was clarified and effects of Neuritin on ligand JAG1 protein expression, endocytosis, and ubiquitination were examine in Notch signaling pathway.Methods:Kidney 293 Cells were transiently transfected with the pIRES-HA-JAG1 plasmid, pcDNA3.1-flag-neuralized plasmid, pcDNA3.1-his-neuritin plasmid and PBS/U6-neuritin siRNA plasmid vector using Lipofectamine 2000. Overexpression of Neuritin, Expression level of JAG 1 and Neuralized was examined using immunoblotting; The encytosis of JAGI effected was observ-ed by Fluorescence microscope; Knock down Neuritin, expression level of JAG 1 effected was examined by using immunoblotting; The encytosis of JAG1 effected was observed by Fluorescence microscope. Finally, ubiquitination of ligand JAG1 effected by Neuralized and Neuritin was observed by Ubiquitination assay.Results:1.Completeness and correctness of the required plasmid by PCR and gene sequencing technology;2. Expression level of JAG 1 and Neuralized was examined by using Immunoblotting:(1) Overexpression of Neuritin increased the expression of JAG1, and decreased the expression of Neuralized;(2) Knock down Neuritin decreased the expression of JAG1, and increased the expres-sion of Neuralized.3. The encytosis of JAG1 was observed by Fluorescence microscope:(1) Overexpression of Neuritin decreased the endocytosis of JAG1;(2) Knock down Neuritin increased the endocytosis of JAG 1.4. Ubiquitination of ligand JAG1 was observed by Ubiquitination assay:Overexpression of Neuritin decreased the ubiquitination of JAG1.Conclusion:1.Neuritin can increase the expression of JAG1, and can decrease the expression of Neuralized;2.Neuritin can decrease the endocytosis of JAG 1;3.Neuritin can decrease the ubiquitination of JAG1.