Figure 4.

As(III)- and MMA(III)-induced activation of Nrf2 is independent of C151 in Keap1. (A) The ARE luciferase reporter gene assay was performed in the same way as described in Figure 1A, except that Keap1-C151S, rather than Keap1-WT, was cotransfected in half of the samples. The transfected cells were treated for 16 h with 10 µM As(III), 1 µM MMA(III), 50 µM tBHQ, or 7.5 µM SF prior to the measurement of firefly and renilla luciferase activities. (B) The same lysates from A were used for immunoblot analysis with anti-HA, anti-Keap1, and anti-α-tubulin antibodies. (C) In vivo ubiquitination assay was performed in MDA-MB-231 cells cotransfected with expression vectors for HA-ubiquitin and Gal4-Neh2, and an expression vector for either Keap1-WT or Keap1-C151S. The transfected cells were treated for 4 h with H₂O, 20 µM As(III), 2 µM MMA(III), or 100 µM tBHQ, along with 10 µM MG132. The cells were lysed and proteins were denatured by heating. The Gal4-Neh2 proteins were immunoprecipitated with anti-Nrf2 and immunoblotted with anti-HA for detection of ubiquitin-conjugated Gal4-Neh2 proteins (top panel). Small aliquots of samples were subjected to immunoblot analysis with anti-Gal, anti-Keap1, and anti-α-tubulin antibodies (bottom three panels).