Figure 3

A transactivation domain is located in the VWC module of the CCN3 protein. A) Nomenclature and schematic depicting of the modular structure of the CCN3 protein. IGFBP: Insulin Like Growth Factor Binding Protein-like module; VWC: Von Willebrand factor-like module; TSP1: Thrombospondin-like module; CT: C-terminal module. Acidic residues are colored in blue, proline in red, and are marked by an asterisk in the human CCN3 VWC module. B) The pGBT9 transfectants (Gal4DBD fusion proteins) were selected and grown in minimal medium deprived of tryptophane. The Y190 recipient yeast used in this experiment contains a recombinant lacZ reporter gene cloned downstream a promoter containing upstream Gal4 DBD binding sites (activator binding sites). Qualitative assays were performed on Whatman filter paper. The β-galactosidase activity was monitored every half hour for a total period of 8 hours. C) Upper panel: diagram depicting the mammalian reporter system used in this study (pFA/pFR-Luc, Stratagen). Lower panel left: Immunocytofluorescent detection of Gal4DBD-NH3 protein in transiently transfected BHK21 cells, showing nuclear localization of the fusion protein using anti-His antibody (protocol for immunodetection described in [5]). Lower panel right: BHK21 cells were co-transfected with 1 μg of pFR-Luc and increasing amounts of NH3. The total amount of DNA used for each transfection was kept constant by adjustment with pFA DNA. BHK21 (baby hamster kidney 21) cells were grown and transfected as decribed in [5]. Posttranfection (48 h), luciferase and β-galactosidase activity from cell lysates was measured as described in [5].