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. 2005 Jan;25(1):162–171. doi: 10.1128/MCB.25.1.162-171.2005

FIG. 2.

FIG. 2.

KEAP1 promotes NRF2 degradation. (A) Schematic illustration of NRF2 protein and mutations characterized in this study. BZIP, basic region leucine zipper; aa, amino acids. (B) 293T cells were cotransfected with the plasmids expressing HA-tagged KEAP1 (HA-KEAP1) and Myc-tagged NRF2 (Myc-NRF2) (wild-type [WT] or mutant NRF2). Twenty hours after transfection, cells were treated with MG132 (25 μM) for 4 h prior to lysis, and the NRF2-KEAP1 association was examined by IP-Western. IB, immunoblotting; α-Myc, anti-Myc antibody; α-HA, anti-HA antibody. (C) HeLa cells were cotransfected with the plasmids expressing HA-tagged KEAP1 and FLAG-tagged wild-type or mutant NRF2. Twenty-four hours after transfection, cells were lysed, and lysates (0.1 mg) were resolved by SDS-PAGE, followed by immunoblotting (IB) with anti-FLAG (α-FLAG) or anti-HA antibodies. (D) GFP-tagged NRF2 was transfected to HeLa cells either alone or with the plasmid expressing DsRed-tagged KEAP1. Twenty-four hours after transfection, the levels of expression of GFP-NRF2 and DsRed-KEAP1 were examined by fluorescence or phase-contrast microscopy.