FIG. 8.

Keap1 confers postinduction repression of Nrf2 by escorting nuclear export of Nrf2. (A and B) Postinduction repression of the steady-state levels of Nrf2 was accessed in MDA-MB-231 cells cotransfected with an expression vector for Nrf2 and an expression vector for either Keap1-WT or Keap1-NES. Cells were treated with 100 μM tBHQ for 4 h. After removal of tBHQ by washing, cells were further incubated in normal medium for the indicated time periods. Total cell lysates were subjected to immunoblot analysis with anti-HA, anti-CBD, and anti-lamin A antibodies. (B) The relative intensities of the Nrf2 bands were quantified by the ChemiDoc XRS gel documentation system from Bio-Rad and plotted on a linear graph. (C) Postinduction repression of the Nrf2-dependent transcriptional activity was determined in MDA-MB-231 cells cotransfected with plasmids encoding an ARE-firefly luciferase, thymidine kinase-Renilla luciferase, Nrf2, and the indicated Keap1 protein. The transfected cells were exposed to 50 μM tBHQ for 16 h. Following removal of tBHQ, cells were further incubated in normal medium for the indicated time periods prior to measurement of firefly and Renilla luciferase activities. The experiment was repeated three times, and standard deviations are shown as error bars. Untreat, untreated.