FIG. 6.

Cul3 promotes Nrf2 polyubiquitination. (a) Lysates were collected from 293T cells that were mock treated (lane 2) or treated with 10 μM MG132 (lane 3) for 2 h. Nrf2 was detected via immunoprecipitation followed by immunoblot analysis. Lane 1 shows a control precipitation. (b) 293T cells were transfected with plasmids encoding 6xHis-ubiquitin, Nrf2, Myc-Keap1, Flag-Cul3, and Cul3N418 in the indicated combinations. Cells were lysed under denaturing conditions, and ubiquitin-containing complexes were affinity purified and resolved via SDS-PAGE. The presence of Nrf2, Cul3, and Keap1 was determined via immunoblot analysis.