The Study Of Sumoylation On PML-NBs

PML-NBs is a subnuclear structure of mammalian cell nucleus called ND (nuclear domain)-10, Kremer nuclear and PML oncogene domain. It is found out during recent ten years that PML plays great roles in almost all biological functions such as transcription regulation, DNA repair, cell apoptosis, cell sentence and cardiavascular formation. In the process of PML-NBs formation and degradation, many located with PML-NBs play functions, and SUMO modification is important. SUMO is a small protein like ubiquitin in recent years found in all eukaryotic, it pays vital roles in protein localization, chromatin structure maintenance, transcription regulation, DNA repair, nuclear and cytoplasm transport, signal transduction.First,based on the rabbit anti human PML polyclonal antibodies (pAb), the specificity of the pAb was detected through immunofluorescence:Hela cells were transient transfected with pCMV-Myc-PML, after comparison with santacruz monoclonal PML antibody (mAb), the pAb has high specificity.Secondly, the colocalization of PML-NBs with SUMO-1/2 and the modification of PML by SUMO-1/2 were got by immunofluorescence and western blot. (1)Hela cells were transient transfected with pCMV-HA-PML/pCMV-Myc-SUMO-1, pCMV-HA-PML/pCMV-Myc- SUMO-2, the subcellular localization of PML-NBs and the colocalization of PML-NBs and SUMO-1/2 were obtained by immunofluorescence. (2)Hela cells were transient transfected with pCMV-Myc-PML/pYFP-C1-SUMO-1, pCMV-Myc-PML/pYFP-C1-SUMO-2, western blot result showed that the modifications of SUMO-1/2 on PML were different.Further, Hela cells were synchronized at different phases of cell cycle by two medicines, it is found that there are differences of the colocalization of PML and SUMO-1/2.Finally, Hela cells were transient transfected by pGFP-C1-PML/pDsRed-C1-RNF4, pGFP-C1-SUMO-1/pDsRed-C1-RNF4, pGFP-C1-SUMO-2/pDsRed-C1-RNF4, it is found that they colocalize.