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Updated: May 12, 2026
Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM)
Published on: December 22, 2015
[Identification of interaction between BPHL and PML-C]
Chen Chu1, Bei-Zhong Liu, Liang Zhong
1Central Laboratory of Yongchuan Hospital, Chongqing Medical University, Chongqing 402160, China.
Objective:
To explore the interaction between BPHL and PML-C by co-immunoprecipitation and yeast two-hybird system.
Methods:
The recombination expression plasmids pGBKT7-PML-C and pACT2-BPHL were cotransformed into yeast AH109, to investigate their interaction in vivo. The expression vector of HA-tagged fusion protein (pCMV-HA-PML-C) and the expression vector of myc-tagged fusion protein (pCMV-myc-BPHL) were constructed and identified respectively, and cotransfected into human embryo kidney 293 (HEK293) cells. The interaction between PML-C and BPHL was investigated by co-immunoprecipitation in vitro.
Results:
Blue clones were found in QDO/5-bromo-4-chloro-3-indolyl-alpha-D-galactoside (X-alpha-gal) plate, eukaryotic expression vectors named as pCMV-HA-PML-C and pCMV-myc-BPHL were constructed and confirmed with double restriction enzyme digestion and co-transfected into HEK 293 cells successfully. After immunoprecipitation of HA-PML-C with anti-HA polyclonal antibody, expressed myc-BPHL protein was identified by Western blot with anti-c-myc monoclonal antibody from immunoprecipitated complex.
Conclusion:
The eukaryotic expression vector of PCMV-HA-PML-C and PCMV-myc-BPHL were constructed successfully. The interaction between PML-C and BPHL was identified by co-immunoprecipitation and yeast two-hybird technique.
Insights
This study demonstrates a direct interaction between PML-C and BPHL proteins using both yeast two-hybrid and co-immunoprecipitation assays. These findings confirm the physical association of PML-C and BPHL, advancing our understanding of their cellular roles.
Area of Science:
- Molecular Biology
- Protein-Protein Interactions
Background:
- The study investigates the molecular mechanisms underlying cellular processes involving PML-C and BPHL.
- Understanding protein interactions is crucial for elucidating complex biological pathways.
Purpose of the Study:
- To confirm and characterize the interaction between PML-C and BPHL.
- To establish a foundation for further research into the functional significance of this interaction.
Main Methods:
- Co-transformation of yeast with recombination expression plasmids (pGBKT7-PML-C and pACT2-BPHL).
- Construction and identification of eukaryotic expression vectors for HA-tagged PML-C and myc-tagged BPHL.
- Co-transfection of HEK293 cells with expression vectors.
- In vitro co-immunoprecipitation assays using anti-HA and anti-c-myc antibodies.
Main Results:
- Successful construction and verification of eukaryotic expression vectors pCMV-HA-PML-C and pCMV-myc-BPHL.
- Identification of blue clones in yeast, indicating interaction in vivo.
- Confirmation of protein interaction in vitro via co-immunoprecipitation, with myc-BPHL detected in the immunoprecipitated complex of HA-PML-C.
Conclusions:
- The study successfully constructed the necessary eukaryotic expression vectors.
- Both yeast two-hybrid and co-immunoprecipitation techniques confirmed a direct interaction between PML-C and BPHL.
