摘要
目的 :发现调控Sonic Hedgehog(Shh)信号转导的泛素连接酶。方法 :第一轮筛选,利用荧光素酶报告基因(8×GliBS-Luc)检测系统,检测相应HECT家族E3泛素连接酶的siRNA对Shh信号通路活性的影响;第二轮筛选,利用细胞免疫荧光激光共聚焦检测系统,检测上述siRNA对Shh的受体Ptch1蛋白原纤毛定位的影响。综合2轮筛选结果,初步发现影响Shh信号通路活性的HECT家族E3泛素连接酶。结果:通过2轮筛选,发现Smurf1、Smurf2、Ube3c、Wwp1、Wwp2共5个泛素连接酶,不仅能调节Ptch1蛋白的原纤毛定位,也可以增加通路下游转录因子Gli的活性。结论:建立了筛选调控Shh信号通路的泛素连接酶的平台。在初步筛选的27个HECT E3泛素连接酶中,有5个成员参与调控Shh信号通路。
Objective：Screening for HECT E3 ligases that can regulate Sonic Hedgehog （Shh） signaling pathway. Methods：In the first round of screening,siRNAs of the HECT E3 ligases were tested for Shh pathway transduction by GliBS-luciferase assay. And in the second round,these siRNAs were tested for the localization of Patchedl-GFP in primary cilia by immunofluorescence staining. Results： After two rounds of screening, 5 HECT E3 ligases were identified to regulate Shh pathway, which are Smurfl, Smurf2, Ube3c, Wwpl and Wwp2. Conclusion：A screening system of new regulators of Shh signaling was setup,and 5 HECT E3 ligases were found from our preliminary screening.
出处
《南京医科大学学报（自然科学版）》
CAS
CSCD
北大核心
2013年第6期715-721,共7页
Journal of Nanjing Medical University(Natural Sciences)
基金
教育部博士学科点基金(20103234110003)
国家自然科学基金国际(地区)合作与交流项目(81261120386)
国家自然科学基金青年基金项目(81101497)
