title: [Construction and identification of eukaryotic expression vectors of IKKα carrying Ser176/180 phosphorylation site mutants].
abstract: OBJECTIVE
  To construct eukaryotic expression vectors of the inhibitor of nuclear factor Kappa-B kinaseα (IKKα) containing Ser176/180 phosphorylation site mutants.
  
  
  METHODS
  Using PCR, we amplified two IKKα gene fragments, one containing Ser176/180 site mutants and non-sense mutation KpnI and the other with non-sense mutation KpnI. They were subjected to double enzyme digestion, HindIII/KpnI and KpnI/EcoRI, respectively, and then subcloned into pEGFP that had also digested with HindIII/EcoRI beforehand. The location of pEGFP-IKKα (kinase activity, KA)/(kinase death, KD) fusion protein in the eukaryocytes and their effects on P65 nuclear translocation were observed under a confocal microscope. The fusion protein expressions were detected by Western blotting.
  
  
  RESULTS
  Sequencing analysis verified the fidelity of all plasmid DNA constructs. PEGFP-IKKα KA was mainly located in nucleus and cytoplasm, and regulated P65 nuclear shuttling. In contrast, pEGFP-IKKα KD was mostly localized in cytoplasm, and did not initiate P65 nuclear translocation. Western blotting confirmed the expressions of the fusion proteins.
  
  
  CONCLUSIONS
  The eukaryotic expression vectors pEGFP-IKKα KA/KD were successfully constructed and well expressed in the eukaryocytes. The fusion proteins were located in different parts of the cells and exerted different effects on P65 nuclear translocation.
authors.affiliation: Laboratory of Cell Pathobiology, Ministry of Education Key Laboratory of Medical Cell Biology, Department of Cell Biology, College of Basic Medical Science, China Medical University, Shenyang 110001, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: He
authors.affiliation: Laboratory of Cell Pathobiology, Ministry of Education Key Laboratory of Medical Cell Biology, Department of Cell Biology, College of Basic Medical Science, China Medical University, Shenyang 110001, China.
authors.forename: Qianqian
authors.initials: Q
authors.lastname: Zheng
authors.affiliation: Laboratory of Cell Pathobiology, Ministry of Education Key Laboratory of Medical Cell Biology, Department of Cell Biology, College of Basic Medical Science, China Medical University, Shenyang 110001, China.
authors.forename: Yaqin
authors.initials: Y
authors.lastname: Zhu
mesh_terms: D021581:Active Transport, Cell Nucleus; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D051550:I-kappa B Kinase / Q000235:genetics*; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D012694:Serine
chemical_list: D011993:Recombinant Fusion Proteins; D012694:Serine; D051550:I-kappa B Kinase