Identification of Interaction Between BPHL and PML-C

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-α-D-galactoside (X-α-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.