Figure 6. Effect of epoxomicin on TCDD-induced degradation of the AHR and induction of CYP1A1.

A) Hepa-1 or human RPE cells were treated with 0.05% DMSO or epoxomicin (1μM or 5μM) for 1 hr at 37°C. Cells were then exposed to TCDD (2nM) for an additional 4 hrs and total cell lysates prepared. Equal amounts of total cell lysates were resolved by SDS-PAGE, blotted and stained with A-1A anti-AHR IgG (1.0μg/ml), anti-ß-actin IgG (1:1000) or anti-CYP1A1 IgG (1:1000). Reactivity was visualized by ECL with GAR-HRP (1:10,000). B) The level of AHR protein was normalized to the level of actin as detailed [29, 30]. Three independent experiments for each cell line were then averaged and plotted as the mean +/- SE with the DMSO treated cells in each experiment set to 100%. * = statistically different from DMSO treated cells (p<0.001). ** = statistically different from TCDD treated control cells (p<0.001). C) Hepa-1 cells were grown on glass coverslips and treated with DMSO (0.05%), MG-132 (7.5μM), epoxomicin (5μM), or proteasome inhibitor 1 (20μM) for 1 hr at 37°C. Cells were then incubated with t-BOC-L-leucyl-L-methionine (10μM) for 20 minutes and wet mounted on slides in phosphate buffered saline. Fluorescence was observed at 405nm and individual fields photographed for identical times. Bar in the control panel = 5μm.