Figure 6.

Eugenol inhibits Nrf2 ubiquitination in 293 cells. Plasmids expressing Nrf2 and HA-Ub were first co-transfected into 293 cells for 24 h. Cells were subsequently treated with MG132 (2 µM) in the absence or presence of eugenol (50 µg/ml) for 6 h. Whole-cell extracts were prepared and subjected to Nrf2 protein purification using anti-Flag antibody beads. Precipitates were visualized by western blot analysis using anti-HA antibodies. Experiments were performed in triplicate. Nrf2, nuclear factor erythroid 2-related factor 2; HA, human influenza hemagglutinin; HA-Ub, HA-tagged ubiquitin; IP, immunoprecipitation.