FIG. 6.

Oxidative/electrophilic stress does not affect formation of the Keap1-Nrf2 complex. Electrophilic modification of Keap1 with biotinylated 15d-PGJ₂ does not affect formation of the Keap1-Nrf2 complex. 293T cells expressing Keap1 were treated with biotinylated 15d-PGJ₂ (Bio-PGJ₂, lanes 1, 4, and 5; 10 μM final concentration) for 1 h. Whole-cell extracts were prepared and mixed with whole-cell extracts of Nrf2-expressing (lanes 1, 3, and 5) or wild-type (lanes 2 and 4) cells. Biotinylated Keap1 was precipitated from the mixed cell extracts using avidin beads, and precipitates were subjected to immunoblot analysis with anti-Nrf2 and anti-Keap1 antibodies (top and bottom panels, respectively).