FIG. 4.

Reduced Cys273 and Cys288 are crucial for the ubiquitin-dependent degradation of Nrf2. (A) Alanine mutation or oxidation of Cys273 and Cys288 impairs Keap1-mediated degradation of Nrf2. The degradation activity of Keap1 was monitored by an in vivo degradation assay. The expression plasmids for Nrf2 and wild type or C273&288A mutant Keap1 (2 μg and 1.5 μg, respectively) were transfected into Cos7 cells, as indicated in the figure. EGFP plasmid (50 ng) was cotransfected to verify the transfection efficiency. At 24 h after transfection, cells were treated with DMSO (lanes 1 to 3 and 5) or tBHQ (lane 4; final concentration, 100 μM) for 12 h. Whole-cell extracts were prepared and subjected to immunoblot analysis using anti-Nrf2 and anti-GFP antibodies (top and bottom panels, respectively). (B) Alanine mutation or oxidation of Cys273 and Cys288 impairs Keap1-mediated ubiquitination of the Neh2 domain. An in vivo ubiquitination assay was performed. A GFP-fused Neh2 domain that harbors the Keap1-dependent ubiquitination site was used in this assay (Neh2-GFP). The expression plasmids for Neh2-GFP and wild-type Keap1 or Keap1-C273&288A mutant were transfected into 293T cells, as indicated in the figure, along with a His-tagged ubiquitin (HisUb) plasmid. At 24 h after transfection, cells were treated with MG132 (final concentration, 2 μM) in the absence (lanes 1 to 3 and 5) or presence (lane 4; final concentration, 100 μM) of tBHQ for 12 h. Whole-cell extracts were prepared and subjected to Ni²⁺ affinity purification. Precipitates were visualized by immunoblot analysis with anti-Nrf2 antibody.