FIGURE 11.

Analysis of nuclear body-enriched preparation reveals that RNF4 induces degradation of Nrf2 in arsenic trioxide-treated cells. Cells were grown and treated as in the legend to Fig. 10. A, whole cell lysate (W) prepared from cultures treated with As₂O₃ (2 μm) for 4 h was separated into cytoplasmic (C) and nuclear body-enriched (NB) fractions as in Fig. 2. RNF4-YFP was detected with anti-GFP antibody (Invitrogen). Keap1 was detected with anti-Keap1 antibody (ab31973; Abcam). B, expressed levels of wild-type RNF4 (RNF4wt-YFP) and mutant RNF4 (RNF4-CS1-YFP) (right panel) and steady-state levels of endogenous Nrf2 (left panel) in the NB fraction. C, RNF4-mediated degradation of endogenous Nrf2, measured in nuclear body-enriched fraction of cells transfected with plasmid encoding wild-type RNF4-YFP. Forty eight hours after transfection, HepG2 cells were incubated with As₂O₃ (2 μm) and cycloheximide (CHX) (50 μg/μl) and then harvested at the indicated time points thereafter. Nuclear body-enriched fractions (NB fractions) were then immunoblotted for Nrf2. Representative blots are shown. The blots were quantified densitometrically using UN-SCAN-IT software (Silk Scientific, Inc., Orem, UT). The values plotted are means ± S.E. for five experiments. The data are presented as percentage plots, taking the values for no treatment with cycloheximide (zero time) as 100%. Open circles, treatment with empty vector; closed squares, treatment with RNF4wt-YFP.