Figure 3.

As(III) and MMA(III) have no effect on the Nrf2-Keap1 complex, but enhance the Cul3-Keap1 interaction. (A) Interaction of endogenous Nrf2 and Keap1 was measured by immunoprecipitation assay in MDA-MB-231 cells treated with H₂O, 20 µM As(III), 2 µM MMA(III), 100 µM tBHQ, or 15 µM SF for 4 h. The Keap1 immunoprecipitates were blotted with anti-Nrf2 and anti-Keap1 antibodies (top two panels). Small aliquots of total lysates were subjected to immunoblot analysis with anti-Nrf2, anti-Keap1, and anti-α-tubulin antibodies (Bottom three panels). (B) Keap1-CBD and HA-Cul3 cotransfected MDA-MB-231 cells were treated similarly with the indicated chemicals. The Keap1 complexes were purified using chitin beads and subjected to immunoblot analysis with anti-HA and anti-CBD antibodies for detection of Cul3 and Keap1 (top two panels). Small aliquots of total lysates were immunoblotted with anti-HA for detecting Cul3, anti-CDB for detecting Keap1, and anti-α-tubulin for loading control (bottom three panels).