摘要
目的:Abba1在体外实验中能介导细胞产生ruffling,其N端的IMD区域中13个赖氨酸为重要的功能氨基酸位点,但Abba1单独的IMD却不足以介导细胞产生ruffling,本研究旨在初步探讨Abba1的C端序列影响细胞产生ruffling的分子机制。方法:先构建Abba1基因重组表达质粒,分别瞬时转染COS7细胞,通过免疫荧光观察细胞形态改变,并用Western blot检测相应GTP-Rac1的蛋白水平变化。结果:过表达全长Abba1的COS7细胞形成ruffling,与对照组相比,细胞中GTP-Rac1蛋白水平升高(P<0.05);过表达Abba1-IMD和Abba1-del-357-722aa-WH2的COS7细胞不形成ruffling,与对照组相比,相应细胞中GTP-Rac1蛋白水平降低(P<0.05)。结论:Abba1的357-722aa这段区域参与介导细胞产生ruffling,而且这种影响与GTP-Rac1蛋白水平的降低有关。
Objective：To study whether Abba1’s C-terminal region might participate in the formation of membrane ruffling.Methods：Several recombinant plasmids,such as pEGFP,pEGFP-hAbba1,pEGFP-hAbba1-IMD,pEGFP-hAbba1-del-357-722aa-WH2 were prepared.COS7 cells were transiently transfected with these plasmids,respectively.Immunofluorescent microscopy was carried out to detect the formation of membrane protrusions.And GTP-bound Rac1（GTP-Rac1）in cell lysates was determined by pull-down with GST-PAK-CRIB followed by Western blot with Rac1 antibody.Results：From immunofluorescent microscopy,unlike overexpression of Abba1 inducing the formation of membrane ruffling,cells overexpressing Abba1-IMD or Abba1-del-357-722aa-WH2 displayed microspikes.Pull-down combined Western blot analysis revealed that the relative level of GTP-Rac1 was increased in the cells with overexpression of Abba1.However,the level was decreased in cells transfected with Abba1-IMD and Abba1-del-357-722aa-WH2.Conclusion：The data indicated that C-terminal region affected Abba1 inducing membrane ruffling in COS7 cells,which was connected with the decrescent level of GTP-Rac1.
出处
《南京医科大学学报（自然科学版）》
CAS
CSCD
北大核心
2011年第8期1110-1114,共5页
Journal of Nanjing Medical University(Natural Sciences)
基金
江苏省卫生厅面上项目(H200912)
