FIG. 5.

ProTα competes with Nrf2 for binding to Keap1 in vitro. (A) Recombinant zz-Keap1 (50 ng) was immobilized on IgG-Sepharose and charged with ³²P-ProTα. Unbound ProTα was removed, and the immobilized Keap1-ProTα complex was incubated with buffer alone (solid line) or with the same buffer containing recombinant Nrf2 (250 ng) (dotted line). At the indicated time intervals, the amount of ³²P-ProTα in the pulled-down fractions was determined. The amount of matrix-bound ProTα at zero time was taken as 100%. (B) Recombinant zz-Keap1 (50 ng) immobilized on IgG-Sepharose was charged with recombinant Nrf2 (250 ng). Unbound Nrf2 was removed, and the immobilized Keap1-Nrf2 complex was incubated in parallel with recombinant wild-type ProTα (lanes 2 and 5), with the MProTα(44,50) mutant (lanes 3 and 6) (50 μg each), or with buffer alone (lanes 1 and 4) for 1 h. The amount of Nrf2 in the pulled-down (Bound to Keap1, lanes 1 to 3) and supernatant (Displaced, lanes 4 to 6) fractions was determined by Western blotting analysis of each fraction with anti-Nrf2 antibody. Lane 7, Nrf2 used as a marker. (C) Displacement of Nrf2 from the Nrf2-Keap1 complex by ProTα is dose dependent. zz-Keap1-Nrf2 complex was formed as in panel B and challenged with increasing amounts of wild-type ProTα for 1 h. Amounts of displaced Nrf2 were determined by immunoblotting as in panel B. Lanes 1 to 4, 0.05, 0.5, 5.0, and 50 μg of ProTα, respectively. Lane 5, Nrf2 used as a marker.