FIG. 5.

Mutant Keap1 proteins increase the stability of Nrf2. (A) COS1 cells were cotransfected with an expression vector for HA-Nrf2 (all lanes) and expression vectors for either wild-type (WT) or mutant Keap1 proteins (lanes 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, and 18). The transfected cells were either untreated (lanes 1, 2, 4, 5, 7, 8, 10, 11, 13, 14, 16, and 17) or treated with 25 μM tBHQ for 16 h (lanes 3, 6, 9, 12, 15, and 18). Cell lysates were electrophoresed through an SDS-7.5% polyacrylamide gel and subjected to immunoblot analysis with anti-HA (α-HA) (top panel) or anti-Keap1 (α-Keap1) (middle panel) antibodies. Equivalent amounts of protein from each sample were also subjected to immunoprecipitation with an anti-Keap1 antibody. The immunoprecipitated proteins were electrophoresed through an SDS-7.5% polyacrylamide gel and subjected to immunoblot analysis with anti-HA (bottom panel). Only the relevant portions of the autoradiographs are shown. The Nrf2 and Keap1 proteins are indicated by arrows. +, present; −, absent. (B) COS1 cells were cotransfected with an expression vector for HA-Nrf2 and expression vectors for either wild-type Keap1 (top panel), Keap1-C151S (second panel from top), Keap1-C273S (third panel from top), or Keap1-C288S (bottom panel). Transfected cells were pulse labeled with media containing [³⁵S]methionine and [³⁵S]cysteine for 30 min and chased with media containing unlabeled methionine for 0, 1, 3, 5, and 7 h. Cell lysates were collected following the indicated chase periods and subjected to immunoprecipitation with anti-HA antibodies. The immunoprecipitated proteins were subjected to electrophoresis through an SDS-7.5% polyacrylamide gel and visualized by fluorography. The data shown are from one experiment. The pulse-chase analysis was repeated a second time with nearly identical results. (C) The relative intensities of immunoprecipitated Nrf2 were quantified by phosphorimager analysis and plotted on a semi-log graph. The amount of Nrf2 present at the end of the 30-min labeling period was set at 1. The value at each subsequent time point is the average of two individual experiments. The half-life of Nrf2 in the presence of the indicated Keap1 proteins is indicated in the inset.