人源RhoA被SUMO2/3修饰。
[Human RhoA is modified by SUMO2/3].
作者信息
Wan Yingcong, Li Chunyan, She Jiayao, Wang Jingya, Chen Ming
机构信息
Department of Neurobiology, Southern Medical University, Guangzhou 510515, China.
出版信息
Nan Fang Yi Ke Da Xue Xue Bao. 2018 Jan 30;38(1):75-80. doi: 10.3969/j.issn.1673-4254.2018.01.12.
OBJECTIVE
To investigate whether human RhoA is modified by SUMO.
METHODS
Overlap extension PCR and double digestion technique were used to construct the eukaryotic expression vector pcDNA3-3flag-RhoA, which was identified by sequencing. The plasmid was transfected into HEK293T cells and its expression was detected by Western blotting. Immunofluorescence assay was used to detect whether RhoA is co-localized with SUMO. Co-Immunoprecipitation was used to detect whether RhoA is modified by SUMO.
RESULTS
The recombinant plasmid pcDNA3-3flag-RhoA was successfully constructed and verified. Western blotting showed that the recombinant plasmid pcDNA3-3flag-RhoA expressed abundant fusion protein in HEK293T cells. Immunofluorescence showed that RhoA was co-localized with SUMO2/3 but not with SUMO1. Co-immunoprecipitation verified that RhoA was modified by SUMO2/3 but not SUMO1.
CONCLUSIONS
Human RhoA is modified by SUMO2/3 and probably participates in the regulation of axon regrowth after nervous system injury.
目的
研究人RhoA是否被小泛素样修饰蛋白（SUMO）修饰。
方法
采用重叠延伸PCR和双酶切技术构建真核表达载体pcDNA3-3flag-RhoA，并进行测序鉴定。将该质粒转染至人胚肾293T细胞（HEK293T细胞），通过蛋白质免疫印迹法检测其表达情况。采用免疫荧光法检测RhoA与SUMO是否共定位。采用免疫共沉淀法检测RhoA是否被SUMO修饰。
结果
成功构建并验证了重组质粒pcDNA3-3flag-RhoA。蛋白质免疫印迹法显示，重组质粒pcDNA3-3flag-RhoA在HEK293T细胞中表达了丰富的融合蛋白。免疫荧光显示，RhoA与SUMO2/3共定位，但与SUMO1不共定位。免疫共沉淀证实，RhoA被SUMO2/3修饰，但未被SUMO1修饰。
结论
人RhoA被SUMO2/3修饰，可能参与神经系统损伤后轴突再生的调控。
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