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


CONCLUSIONS
The interaction between NLS-RARalpha and UBQLN1 can be verified by yeast two-hybrid assay and co-immunoprecipitation.
Zhu
Dan
D
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.
Wang
Chong
C
Liu
Beizhong
B
Wu
Yan
Y
Zhong
Liang
L
Wang
Chunguang
C
D021581:Active Transport, Cell Nucleus; D048868:Adaptor Proteins, Signal Transducing; D000071183:Autophagy-Related Proteins; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals / Q000235:genetics / Q000378:metabolism*; D054730:Protein Interaction Domains and Motifs / Q000502:physiology*; D018168:Receptors, Retinoic Acid / Q000235:genetics / Q000378:metabolism*; D000072482:Retinoic Acid Receptor alpha; D014162:Transfection; D020798:Two-Hybrid System Techniques
D048868:Adaptor Proteins, Signal Transducing; D000071183:Autophagy-Related Proteins; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D019913:Nuclear Localization Signals; C000607071:RARA protein, human; D018168:Receptors, Retinoic Acid; D000072482:Retinoic Acid Receptor alpha; C107792:UBQLN1 protein, human