ABSTRACT
Objective: To obtain monoclonal antibodies against programmed cell death 10(PDCD10) for further study of the structure and function of PDCD10 protein.Methods: Balb/c mice were immunized with recombinant PDCD10,hybridoma cell lines secreting monoclonal antibodies against PDCD10 were screened by regular cell fusion and subcloning approach.The specificities of these monoclonal antibodies were determined by ELISA,Western blotting and Immunofluorescecence assay.Results: Three hybridoma cell lines(5G1,4F7 and 3H5) stable in secreting specific monoclonal antibodies were successfully obtained.Subclass of IgG belonged to IgG1(4F7 and 5G1)and IgG2b(3H5),respectively.The ascite titers of these monoclonal antibodies reached 1∶10~7.They could specifically bind to recombinant PDCD10 and endogenous and overexpressed PDCD10 proteins proved by ELISA and Western blotting.They failed to react with E.coli lysates and glutathione S-transferase(GST).In addition,these three monoclonal antibodies could recognize different epitopes of PDCD10 proteins assessed by immune fluorescence competitive binding assay.Both endogenous and overexpressed PDCD10 protein mainly located in the nucleus.Conclusion: Monoclonal antibodies against PDCD10 with high titers and specificity have been successfully prepared,which has laid the foundation for further study of PDCD10 protein.
ABSTRACT
Mitochondria are involved not only in energy metabolism but also in free radical metabolism. Superoxide anion can be generated through a way of electron leak of respiratory chain and the reactive oxygen species (ROS ) can be formed in the further reactions of O2* in mitochondria. The role of mitochondria in anti-oxidant functions and cell apoptosis is discu ssed in terms of electron leak of respiratory chain, uncoupling of oxidative pho sphorelation, mitochondrial pore, Box- or/and PTP-mediated release of cytochro me c from mitochondria and so on. The signaling act of ROS is emphasized in the regulation of cell apoptosis.