Fig. 2

Electromobility shift and supershift assays of NFE2 and AREs. Nuclear extracts were prepared from livers of wild-type mice at the indicated time points after dosing with D3T. Synthetic NFE2 and AREs from mGST Ya and hNQO1 were end-labeled and incubated with 5 µg nuclear extract protein. (A) Electromobility shift assays of NFE2 with hepatic nuclear extract isolated 2, 6, 24, and 48 hr after treatment with D3T (0.5 mmol/kg) or 6 hr after vehicle. Competitive binding assays were performed by adding 100-fold excess cold NFE2, hNQO1 and mGST Ya AREs to the reaction mixture containing nuclear extract isolated 48 hr after treatment. (B) Electromobility supershift assays of NFE2 and AREs from mGST Ya and hNQO1. Antibody reacting with the C-terminal of Nrf2 was incubated with DNA and nuclear extracts for 1 hr and then analyzed on 4% acrylamide gels. Nuclear extracts were prepared from four pooled livers and three supershift assays were performed with each DNA probe.