FIG. 5.

Oxidative/electrophilic stress does not cause dissociation of the Keap1-Nrf2 complex. (A) The effects of in vivo tBHQ treatment on the association between Keap1 and Nrf2 were examined by immunoprecipitation. Expression plasmids for Flag-tagged Nrf2 and Keap1 (2 μg and 1.5 μg, respectively) were transfected into 293T cells. At 24 h after transfection, cells were treated with tBHQ (lane 4, 100 μM final concentration) or the proteasome inhibitor MG132 (lane 5, 2 μM final concentration) for 12 h. Whole-cell extracts were prepared and subjected to immunoprecipitation experiments using anti-Flag antibody beads. Immunoprecipitates were visualized by immunoblot analysis with anti-Keap1 and anti-Nrf2 antibodies (top and middle panels, respectively). The expression levels of Keap1 in whole-cell extracts were monitored by immunoblot analysis with an anti-Keap1 antibody (bottom panel). (B and C) In vitro tBHQ treatment does not cause dissociation of the Keap1-Nrf2 complex. Transfection was performed as described above, and whole-cell extracts were prepared from the transfected 293T cells. The expression levels of Nrf2 and Keap1 mutant were monitored by immunoblot analysis using anti-Nrf2 and anti-Keap1 antibodies (B, top and bottom panels, respectively). Whole-cell extracts were treated with DMSO (C, lanes 4 and 8) or tBHQ (lanes 5 and 9, 10 μM final concentration; lanes 6 and 10, 50 μM final concentration; lanes 7 and 11, 500 μM final concentration) for 4 h at 4°C and subjected to immunoprecipitation with anti-Flag antibody beads. Precipitates were visualized by immunoblot analysis using anti-Keap1 and anti-Nrf2 antibodies (top and bottom panels, respectively). Lanes 1 to 3 were loaded with the immunoprecipitates of cells transfected with empty, wild-type Keap1, and C273&288A mutant Keap1 expression vectors, respectively.