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

FIG. 1.

FIG. 1.

NRF2 is rapidly ubiquitinated and degraded by proteasomes. (A) 293T cells were transfected with the plasmid expressing FLAG-tagged NRF2 (NRF2-FLAG). Twenty-four hours after transfection, cells were treated first with MG132 (25 μM) or dimethyl sulfoxide (DMSO) for 5 h and then with cycloheximide (CHX) (75 μg/ml) for periods of time ranging from 10 min to 6 h. Cells were lysed, and 50 μg (lane 1) or 100 μg (lanes 2 to 7) of total lysates were resolved by SDS-PAGE, followed by immunoblotting (IB) with anti-FLAG (α-FLAG) or antiactin (α-Actin) antibodies. (B) 293T cells were transfected with the indicated plasmids expressing FLAG-tagged NRF2 and HA-tagged ubiquitin (HA-Ub). Twenty hours after transfection, cells were treated with MG132 (25 μM) for 2 h prior to cell lysis. Cells were lysed in a SDS lysis buffer and boiled for 15 min. Lysates were then diluted with NP-40 lysis buffer and immunoprecipitated (IP) with anti-FLAG antibody. The washed immunoprecipitates and 50 μg of lysates were resolved by SDS-PAGE, followed by immunoblotting with anti-HA and anti-FLAG antibodies, respectively.