FIG 4.

The effect of Ser550 phosphorylation of Nrf2 on nuclear localization. (A) Nuclear fraction (NF) or cytoplasmic fraction (CF) Nrf2 levels. HEK293 cells were transfected with the plasmid encoding Myc-tagged WT-Nrf2 or the site-directed mutant (S550A-Nrf2 or S550E-Nrf2) and were treated with AICAR for 6 h. (B) Alignment of the NES motif in Nrf2 with the consensus sequence. (C) Immunoblottings for Nrf2 in the nuclear fraction (NF) or cytoplasmic fraction (CF) of HEK293 cells transfected with Myc-tagged WT-Nrf2, S550A-Nrf2, or S550E-Nrf2 for 24 h. (D) The effect of leptomycin B (LMB) on the activation of Nrf2 by AICAR. HepG2 cells were treated with AICAR and/or leptomycin B (10 nM) for 3 h. (E) Immunoblottings for Nrf2 in HEK293 cells treated with leptomycin B for 6 h after transfection with Myc-tagged S550A-Nrf2 or S550E-Nrf2. (F) Protein stability experiment using cycloheximide (CHX). HEK293 cells were treated with 10 μg/ml CHX for the indicated times after transfection with Myc-tagged WT-Nrf2, S550A-Nrf2, or S550E-Nrf2. Data represent means ± SEM of results from three independent experiments (significantly different at the respective times from WT-Nrf2 results [*, P < 0.05] and from S550A-Nrf2 results [#, P < 0.05; ##, P < 0.05]). Con, control. (G) Activation and subcellular localization of AMPKα. Each protein was measured in the subcellular fractions of HepG2 cells treated with AICAR and/or leptomycin B for 30 min. For panels A, C, and E, relative band intensities were compared to those of the respective controls of each construct.