Figure 5
LMP1 + LMP1 BiFC and NF-κB Activity. Fluorescence complementation between the indicated LMP1 combinations was determined. BiFC was determined by flow cytometry as described above (Figure 3). Representative YFP histograms of 1 × 104 mCherry positive cells for LMP1-NYFP +LMP1-CYFP (blue), NYFP-CTAR1/2 + 1-187-CYFP (green), and mCherry alone (no BiFC plasmids, red) are displayed. Localization of LMP1-NYFP +LMP1-CYFP BiFC (panel B) was determined as in Figure 4. Perinuclear (white arrows) and membrane (white arrowheads) BiFC is indicated. Promoter reporter assays were performed by transfection of 293T cells with control pRL-SV40, pNF-κB-luc, and vector (DNA3), LMP1, LMP1-BiFC (LMP1-NYFP + LMP1-CYFP) or A5Y384G BiFC (A5-Y384G-NYFP + A5-Y384G-CYFP) plasmids. Forty hours post-transfection, cells were harvested and dual-luciferase assays were performed. Relative luciferase activity was determined by the firefly luciferase activity of the NF-κB reporter construct relative to the control Renilla luciferase activity. Each combination was performed in triplicate and the mean relative NF-κB activity is displayed. Error bars represent the standard deviations from the mean and the experiment has been repeated three times. In parallel BiFC was determined from the cells in the reporter assay (Panel D) as described above (Figure 3). Representative YFP histograms for DNA3 (red), LMP1 (green), LMP1 BiFC (blue), and A5Y384G BiFC (purple) are displayed.