Figure 3. Western blot and bioluminescence analysis demonstrating expression of Gluc1-AR and JunD-Gluc2 and reconstitution of Gaussialuciferase activity following androgen stimulation in transfected Hep3B cells.

A Schematic diagrams for Gluc1-AR and JunD-Gluc2 fusion constructs.

B. Representative Western blot of Hep3B cell lysates analyzed using AR antibody. Lane (1): Hep3B cells transfected with control vector (pcDNA3.1); Lane (2): control untransfected Hep3B cells; Lane (3): Hep3B cells transfected with Gluc1-AR.

C. Representative Western blot of Hep3B cell lysates analyzed using Gaussia luciferase antibody. Lane (1): Hep3B cells transfected with control vector (pCI); Lane (2): control untransfected Hep3B cells; Lane (3): Hep3B cells transfected with JunD-Gluc2. Membranes were stripped and probed with monoclonal antibody against β-actin to control for protein loading (B,C). Positions of molecular size markers 148 kDa (B) and 98 kDa (C) are shown on the right. Cell lysates were obtained and analyzed by western blot from six independent transfection experiments for each construct with similar results.

D. Gaussia luciferase activity in co-transfected cells: Hep3B cells were co-transfected with Gluc1-AR and JunD-Gluc2, then treated with androgen (2nM R1881) (gray bar) or left untreated (−R1881) (black bar). Cell lysates were collected after 48h and bioluminescence activity of Gaussia luciferase was assayed by measuring light emitted from reconstituted Gluc1-Gluc2 at 480nm. Reconstitution of Gluc1-Gluc2 and resulting Gaussia luciferase activity was significantly increased >5-fold in androgen treated cells compared to untreated control cells. Lysates used for these studies were collected from six independent transfections each run in triplicate. * P < 10⁻⁸.