Figure 4

Western blots showing the effects of varying concentrations of B8 on IκB α. Mononuclear cells were prepared as per methods, plated on 6-well plates, allowed to adhere for 1 h then treated with varying concentrations of B8 (1 μM, 10 μM, 20 μM, 100 μM), gliotoxin (0.1 μg/ml), buffer or a DMSO vehicle control (0.2%) for 30 min at 37°C. After this interval LPS (10 ng/ml) or buffer was added to appropriate wells and left to incubate for a further 45 min. Lysates were prepared, total protein determined and 24 μg of protein per well was run on a 12% SDS-PAGE gel, transferred to PVDF. Blots were blocked before probing with rabbit IκB-α diluted 1:2500, incubated overnight at 4°C. Subsequently, the blots were washed and incubated with goat anti-rabbit HRP, also diluted 1:2500, then developed using standard ECL. Blots are representative of at least 6 similar experiments.