FIG. 6.

The Keap1-C151S protein blocks stabilization and subsequent nuclear translocation of Nrf2 by chemical inducers. Nuclear and cytoplasmic proteins were isolated from MDA-231 cells cotransfected with an expression vector for HA-Nrf2 (all lanes) and expression vectors for either wild-type (WT) Keap1 (lanes 4 to 6), Keap1-C151S (lanes 7 to 9), or Keap1-C273S (lanes 10 to 12). The transfected cells were either untreated (lanes 1, 4, 7, and 10), treated for 16 h with 25 μM tBHQ (lanes 2, 5, 8, and 11), or treated for 16 h with 4 μM sulforaphane (lanes 3, 6, 9, and 12) prior to isolation of nuclear and cytoplasmic proteins. Aliquots of nuclear and cytoplasmic proteins derived from equivalent numbers of cells were electrophoresed through an SDS-7.5% polyacrylamide gel and subjected to immunoblot analysis with anti-HA antibodies. +, present; −, absent.