Notes: The authors used a yeast two-hybrid system to identify proteins that interact with the autoimmune regulator (AIRE) protein. DAXX, a multifunctional protein involved in apoptosis and transcription regulation, interacts with AIRE, as shown through coimmunoprecipitation and colocalization studies. Colocalization of AIRE and DAXX in HeLa cells was demonstrated by confocal microscopy using a Monster Green^® Fluorescent Protein-AIRE fusion protein and endogenous DAXX, which was detected using an anti-DAXX primary antibody and an anti-rabbit secondary antibody conjugated with Texas Red fluorophore. However, AIRE and DAXX did not interact in vitro in a GST pull-down assay using a GST-AIRE construct, radiolabeled DAXX protein expressed in a TNT^® system, and MagneGST™ Glutathione Particles, leading the authors to speculate that the interaction is weak or there are scaffold proteins required for protein interaction. To examine the effect of DAXX on AIRE transcriptional activity, the authors transfected COS-1 and HeLa cells with AIRE and DAXX expression constructs and a luciferase reporter plasmid with the human insulin promoter, then performed Dual Luciferase^® Reporter Assays. AIRE induced transcription of the insulin promoter, but coexpression of DAXX suppressed this transcriptional activation. (4153)

Notes: These authors used the Flexi^® Vector System to prepare ORF clones encoding 1929 human genes and to transfer a subset of these clones to various expression vectors for further analysis. They created HaloTag^® fusion proteins and examined expression of these proteins in vitro and in COS7 and HEK293 cells. They also performed comparisons between the Flexi^® System and Gateway^® cloning system, specifically examining the effects of flanking sequences on protein expression in in vitro translation systems and confirming that the cellular localization of the HaloTag^® fusion proteins was consistent with results obtained using GFP-fusions. (3800)

Notes: As part of this study, the effect of hepatocyte growth factor (HGF) gene therapy was investigated using a rat model of acute myocarditis. The HGF gene was transfected into cardiac myocytes using a hemagluttinating virus of Japan (HVJ)-envelope vector kit. HVJ-envelope vector containing the hMGFP gene from the Monster Green^® phMGFP Vector was used as a transfection control. (3349)

Notes: A fusion protein construct was made using the Monster Green™ Fluorescent Protein phMGFP Vector and the PCR-amplified Open Reading Frame E (ORF-E) from Bovine herpesvirus 1. The construct was transfected into mouse neuroblastoma (Neuro-2A) cells (ATCC CCL131), human neuroblastoma (SK-N-SH) cells, rabbit skin cells and bovine kidney cells. Transfected cells were visualized using an Olympus FW500/BX60 confocal microscope with 488nm excitation laser and 522nm emission filter set. The ORF-E-MGFP protein was localized in discreet domains within the nucleus of Neuro-2A and SK-N-SH cells. In rabbit skin cells and bovine kidney cells the ORF-E-MGFP protein was detected in the cytoplasm and nucleus. (3029)