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. 2005 Jan;25(1):162–171. doi: 10.1128/MCB.25.1.162-171.2005

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Copyright © 2005, American Society for Microbiology

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FIG. 6. — KEAP1-CUL3-ROC1 complex ubiquitinates NRF2 in vitro. (A) KEAP1-CUL3-ROC1 complex. 293T cells were cotransfected with the plasmids expressing the indicated proteins, Myc-tagged CUL3 (Myc-CUL3), T7-tagged KEAP1 (T7-KEAP1), and HA-tagged ROC1 (HA-ROC1). Twenty-four hours after transfection, cells were lysed and immunoprecipitated (IP) with anti-Myc antibody (α-Myc). Total lysates (lane 1 and 2) and immunoprecipitates (lanes 3 and 4) were resolved by SDS-PAGE, followed by immunoblotting (IB) with the indicated antibodies. (B) In vitro ubiquitination of NRF2. KEAP1-CUL3-ROC1 complexes were prepared from triply transfected cells by immunoprecipitation using myc antibody and used as the source of E3 ligase. Bacterially expressed and purified FLAG-tagged NRF2 was incubated with KEAP1-CUL3-ROC1 complex, E1, E2, ubiquitin (Ub), and ATP, and reaction mixtures were resolved by SDS-PAGE and immunoblotted with the indicated antibodies.