FIG. 7.

(A) Thirty-five-millimeter-diameter dishes of COS1 cells were cotransfected with expression vectors for Keap1-CBD (0.3 μg, lanes 1, 3, and 4), myc-Rbx1 (0.1 μg, lanes 2 to 4), and the wild-type (WT) or mutant HA-Cul3 proteins (0.3 μg, lanes 2 to 4) as indicated. Cell lysates were analyzed by immunoblotting with the indicated antibodies (bottom three panels) or incubated with chitin beads. Proteins that remained bound to the chitin beads after extensive washing were analyzed by immunoblotting with the indicated antibodies (top two panels). (B) Sixty-millimeter-diameter dishes of COS1 cells were transfected with 0.5 μg each of the expression vectors for HA-Nrf2, Keap1-CBD, myc-Rbx1, and the WT or mutant HA-Cul3 proteins, as indicated in lanes 1 to 4. The transfected cells were treated with MG132 for 5 h prior to cell lysis. Lysates from three 60-mm-diameter dishes were pooled for each sample. One percent of the cell lysates were analyzed by immunoblotting with the indicated antibodies (bottom four panels), and the rest of the lysates were incubated with chitin beads. After washing, the chitin beads were incubated with E1, E2-UbcH5a, ubiquitin, and ATP. The E1 enzyme was omitted from one sample (lane 1). Subsequently, the chitin beads were pelleted and washed, and proteins that were eluted from the beads after boiling under denaturing conditions were immunoprecipitated with anti-Nrf2 (α-Nrf2) antibodies and then analyzed by immunoblotting with antiubiquitin antibodies (top panel). IP, immunoprecipitate; IgG, immunoglobulin G.