核定位信号-视黄酸受体α与泛素连接蛋白1之间的相互作用

Interaction between nuclear localization signal-retinoic acid receptor alpha and Ubiquilin 1.

作者信息

Zhu Dan, Wang Chong, Liu Beizhong, Wu Yan, Zhong Liang, Wang Chunguang

机构信息

Key Laboratory of Laboratory Medicine Diagnostics, Ministry of Education of People's Repulic of China, Faculty of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China.

出版信息

Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2010 Jul;35(7):649-54. doi: 10.3969/j.issn.1672-7347.2010.07.002.

DOI:10.3969/j.issn.1672-7347.2010.07.002

PMID:20693704

Abstract

OBJECTIVE

To identify the interaction between nuclear localization signal-retinoic acid receptor alpha (NLS-RARalpha) and Ubiquilin 1(UBQLN1).

METHODS

The recombination expression plasmids pGBKT7-NLS-RAR and pACT2-UBQLN1, which expressed bait-protein NLS-RARalpha and target protein UBQLN1 respectively, were cotransformated into yeast AH109. The interaction of the expression plasmids in the living cells was investigated by yeast two-hybrid assay. HA-tagged fusion protein (pCMV-HA-NLS-RARalpha) expression vector and Myc-tagged fusion protein (pCMV-Myc-JTV1) expression vector were constructed, identified, and cotransfected respectively into human embryo kidney 293 cells(HEK293). The interaction was detected by co-immunoprecipitation.

RESULTS

Blue clones were found on yeast AH109 plate cotransformated with pGBKT7-NLS-RARalpha and pACT2-UBQLN1. Double restriction enzyme digestion showed that pCMV-HA-NLS-RARalpha and pCMV-Myc-JTV1 were successfully constructed. Then HA-NLS-RAR protein was immunoprecipitated by anti-HA polyclonal antibody and the expression of Myc-UBQLN1 was tested by Western blot with anti-Myc monoclonal antibody from immunoprecipitated complex.

CONCLUSION

The interaction between NLS-RARalpha and UBQLN1 can be verified by yeast two-hybrid assay and co-immunoprecipitation.

摘要

目的

鉴定核定位信号-维甲酸受体α（NLS-RARα）与泛素连接蛋白1（UBQLN1）之间的相互作用。

方法

将分别表达诱饵蛋白NLS-RARα和靶蛋白UBQLN1的重组表达质粒pGBKT7-NLS-RAR和pACT2-UBQLN1共转化至酵母AH109中。通过酵母双杂交试验研究表达质粒在活细胞中的相互作用。构建、鉴定带有HA标签的融合蛋白（pCMV-HA-NLS-RARα）表达载体和带有Myc标签的融合蛋白（pCMV-Myc-JTV1）表达载体，并分别共转染至人胚肾293细胞（HEK293）中。通过免疫共沉淀检测相互作用。

结果

在pGBKT7-NLS-RARα和pACT2-UBQLN1共转化的酵母AH109平板上发现了蓝色克隆。双酶切显示pCMV-HA-NLS-RARα和pCMV-Myc-JTV1成功构建。然后用抗HA多克隆抗体免疫沉淀HA-NLS-RAR蛋白，并用来自免疫沉淀复合物的抗Myc单克隆抗体通过蛋白质免疫印迹法检测Myc-UBQLN1的表达。