The Effect Of Neuritin On The Combination Of Neuralized And Ligand Of Notch Pathway And Its Downstream Gene

Objective: To discuss the effect of Neuritin on the combination of Neuralized and the Notch signalingligand as well as the downstream target genes and the receptor of Notch signaling pathway. Determine theway that Neuritin mediate Notch signaling pathway through Neuralized.Method:（1）Discuss whether Neuritin affect Notch signaling pathway ligand Jag1by mediate Neuralized:observe the change of Jag1expression level on the condition of over-expressing Neuritin and reducingNeuralized with Si-RNA,through immunofluorescence, immune co-precipitation and Western Blottechnique;（2）Detect the quality change of the combination of Jag1and Neuralized under different Neuritinconcentrations: determine the optimal plasmid transfection concentrations and optimal time for Neuralizedcombining through immune co-precipitation and Western Blot, thus we can observe the quality change ofthe combination of Jag1and Neuralized under different Neuritin concentrations.（3）Explore the effect of Neuritin on the expression of Notch intracellular domain (NICD), and thedownstream target gene HES1through co-ImmunoPrecipitation and Western Blot.Results:1.The detection by co-ImmunoPrecipitation and Western Blot suggested that Neuriitn overexpression canincrease the expression of Jag1; After the Neuralized expression was decreased by RNA interference, theexpression of Jag1was significantly increased.2.The concentration-gradient increased Neuriitn can increase the Jag1expression concentration-gradiently,and decrease the affinity of Neuralized and Jag1.3.As with the decrease of the affinity of Neuralized and Jag1, the expression of NICD and Notchdownstream target gene Hes1was decreased simultaneously.Discussion:1.The effect of Neuritin on Notch signaling ligand Jag1was mediated by Neuralized.2.Neuritin suppresses the ubiquitination and endocytosis of Neuralized-mediated Notch signaling pathwayligand Jag1through its effect on the combination of Neuralized and Jag1.3.Neuritin can partly down-regulate the expression of Notch signaling pathway receptor NICD and itsdownstream target gene Hes1.