探讨Abba1的C端序列对COS7细胞产生ruffling的影响
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江苏省卫生厅面上项目(H200912)


C-terminal region affects Abba1 inducing membrane ruffling in COS7 cells
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    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-

    Abstract:

    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.

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陈燕萍,崔伯塔,郑大同,王卓璎,崔晓利,鲁 翔.探讨Abba1的C端序列对COS7细胞产生ruffling的影响[J].南京医科大学学报(自然科学版),2011,(8):1110-1114

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  • 收稿日期:2011-04-27
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