Wan Yingcong, Li Chunyan, She Jiayao, Wang Jingya, Chen Ming
Department of Neurobiology, Southern Medical University, Guangzhou 510515, China.
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.
To investigate whether human RhoA is modified by SUMO.
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.
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.
Human RhoA is modified by SUMO2/3 and probably participates in the regulation of axon regrowth after nervous system injury.
研究人RhoA是否被小泛素样修饰蛋白（SUMO）修饰。
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。
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。
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。
