摘要
目的探索Numb蛋白对α-突触核蛋白(α-synuclein,α-syn)寡泛素化水平的调控。方法分别将EGFP-N1或EGFP-Numb与HA-α-syn共同转染SH-SY5Y细胞;采用细胞免疫荧光法检测α-syn蛋白和Numb蛋白的亚细胞共定位;使用免疫共沉淀的方法检测Numb蛋白与α-syn蛋白的相互结合;利用Western blot检测Numb蛋白对α-syn蛋白寡泛素水平的调控;在MPP+细胞模型中利用细胞免疫荧光检测Numb对α-syn包涵体形成的影响。结果 Numb与α-syn在细胞质中存在亚细胞共定位;Numb蛋白上调α-syn寡泛素化水平;Numb蛋白通过上调α-syn寡泛素水平促进MPP+诱导α-syn阳性包涵体形成。结论 Numb蛋白上调α-syn蛋白寡泛素水平并促进α-syn包涵体形成。
Objeαive To investigate the regulation of Numb on the level of α-synuclein mono-ubiquitination. Methods Transfeαing the recombinant plasmid EGFP-N1, EGFP-Numb, HA-α-synuclein into SH-SY5Y cells with the eu- karyotic cell transfeαion technique. After transfeαed ,we examined colocalization of α-synuclein and Numb by immunofluo- rescence confocal microscopy. And then,we performated co-immmunoprocipitation to examine the interaαion between α-sy- nuclein and Numb. Futhermore,to examine the effeα of Numb overexpression on the level of α-synuclein ubiquitination by Western-blot. Finally,to observe the formation of α-synuclein-positive inclusion bodies in the SH-SY5Ycells treated with 1 mmol/L MPP ＋ by immunostaining. Results In the EGFP-Numb overexpression group,the Numb showed a significant a verlap with o-synuclein,indicating Numb colocalizes with α-synuclein at a subpopulation in cells compared to the control group;SH-SY5Y cells co-transfeαed with EGFP-Numb and HA-α-syn were treated with 10 μmol/L M G132, the lysates were immunoprecipitated and deteαed by Western-blot, revealling that Numb overexpression increased the level of mono- ubiquitinated α-synuclein; In immunostaining of SH-SY5Ycells treated by 1 mmol/L MPP＋ with anti-α-syn antibody, we found α-synuclein aggregates accumulated in the perinuclear area. Conclusions Numb increases the level of mono-ubiquit- ination of α-synuclein and induces the formation of α-synuclein inclusion body.
出处
《中风与神经疾病杂志》
CAS
CSCD
北大核心
2014年第2期100-103,共4页
Journal of Apoplexy and Nervous Diseases
基金
国家自然科学基金(No.81100949)
