FIGURE 10.

Polysumoylated Nrf2 is ubiquitylated by RNF4 resulting in decreased steady-state levels of Nrf2. Where indicated, cells were transfected with 2 μg of vector (pBOS-EFI-H2BGFP) alone or plasmid encoding wild-type RNF4-YFP or mutant RNF4-CS1-YFP. After 48 h, the cells were incubated with or without MG-132 (10 μm) and/or As₂O₃ (5 μm) for 4 h and then harvested. When used together, MG-132 was added 30 min prior to the addition of As₂O₃. Whole cell lysates were immunoprecipitated with anti-Nrf2 antibody (A–C and F–H) or anti-GFP antibody (D and E) and Western blotted as indicated. A–G, nonimmune serum (IgG) was used as control. A and B, Co-IP assays to assess ubiquitylated (A) or sumoylated (B) endogenous Nrf2. C, input control. D and E, Co-IP assays demonstrating interaction of RNF4 with Nrf2. RNF4-YFP was detected with anti-GFP antibody. D, input control. E, interaction of RNF4 with Nrf2. RNF4-YFP. F, wild-type RNF4, but not its mutant, enhances ubiquitylation of polysumoylated Nrf2. G, ubiquitylated Nrf2 is also sumoylated. H, steady-state levels of Nrf2 in cells transfected with plasmid encoding wild-type RNF4-YFP. Cells were treated with As₂O₃ (2 μm) for up to 8 h with or without MG-132 for 8.5 h, lysed, and then Western blotted for Nrf2. The blots were quantified densitometrically using UN-SCAN-IT software (Silk Scientific, Inc., Orem, UT). Values plotted are means for 2–3 experiments. The data are presented as percentage plots, taking the values for no treatment with As₂O₃ (controls) as 100%.