Interaction between nuclear localization signal-retinoic acid receptor α and Ubiquilin 1
10.3969/j.issn.1672-7347.2010.07.002
- VernacularTitle:带核定位信号的维甲酸受体α与Ubiquilin 1蛋白相互作用的验证
- Author:
Dan ZHU
;
Chong WANG
;
Beizhong LIU
;
Yan WU
;
Liang ZHONG
;
Chunguang WANG
- Publication Type:Journal Article
- Keywords:
yeast two-hybrid assay;
co-immunoprecipitation;
protein interaction
- From:
Journal of Central South University(Medical Sciences)
2010;35(7):649-654
- CountryChina
- Language:Chinese
-
Abstract:
Objective To identify the interaction between nuclear localization signal-retinoic acid receptor α (NLS-RARα) and Ubiquilin 1(UBQLN1). Methods The recombination expression plasmids pGBKT7-NLS-RAR and pACT2-UBQLN1, which expressed bait-protein NLS-RARα 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-RARα) 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-RARα and pACT2-UBQLN1. Double restriction enzyme digestion showed that pCMV-HA-NLS-RARα 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-RARα and UBQLN1 can be verified by yeast two-hybrid assay and co-immunoprecipitation.
