Fig. 4.

Nrf2 is acetylated with HDAC inhibition. (A) BEAS2B cells were treated with the proteasome inhibitor MG132 (5 μg/ml) for 1.5 h or with H₂O₂ (75 μM) for 30 min and Nrf2 and lamin C (control) were detected in nuclear extracts by Western blotting. (B) Nrf2 activity was determined in the same extracts using a TransAM kit, (n = 4), (^∗∗p < 0.01 and ^∗∗∗∗p < 0.0001 vs. NT [non-treatment]). (C) BEAS2B cells were stimulated with MG132 (5 μg/ml) for 2 h, with/without TSA (10 ng/ml for 1–2 h). Nrf2 was immunoprecipitated and acetylated-lysine (Ac-K) and HDAC2 were detected with the relevant-antibody. IP: immunoprecipitation, IB: immunoblot, WC: whole cell extracts. IgG: negative control for immunoprecipitation where no cellular lysates where used. The arrow (→) indicates the correct Nrf2 protein size in the whole cell extracts. (D) Band density of acetylated Nrf2 was determined and normalized to total Nrf2 expression (n = 3), ^∗p < 0.05 vs. NT (non-treatment). Keap1 (E) and DJ-1 (F) were immunoprecipitaed from whole cell extracts under the same conditions used in (C).