Figure 1
Engineering of monotransregulators. (A) The activation domains (ADs) of p65 and VP16 proteins were genetically joined through multiple cloning sites (MCS) to the 5′ and 3′ ends, respectively, of the CDC-cDNA to generate the monotransregulator PV. The construct also contains sequences encoding the Flag and 6xHis epitopes at the amino- and carboxyl-termini, respectively (B) MDA-MB-231 cells were infected with the parent recombinant adenovirus (Ad5) at MOI 900, a recombinant adenovirus bearing cDNA for ERα at 50, ERαEBD at 150, PV at 200 and PVEBD at 900 MOI. For all infections, the total MOI was adjusted to 900 by supplementing with the parent Ad5. Extracts (10 µg) of infected MDA-MB-231 cells at 48 h were subjected to Western blotting (WB) using a horseradish peroxidase-conjugated monoclonal Flag antibody Molecular mass in kDa is indicated. NS denotes nonspecific protein detection. (C) Intracellular localization of receptor proteins was examined by immunocytochemistry (ICC). A fluorescein isothiocyanate-conjugated Flag antibody (FITC) localizes constructs to the nucleus stained with 4′,6-diamidino-2-phenylindole (DAPI) at 48 h after infection. (D) Cell extracts (10 µg) at 48 h after infection also were subjected to electrophoretic mobility shift assay (EMSA) with (+) or without (−) a Flag antibody (Ab). ERE specifies unbound and R-ERE denotes receptor-bound radiolabeled ERE. F in panel B indicates the radiolabeled ERE only. In all experiments a representative result from three independent experiments is shown.