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. 2011 May;31(9):1800–1811. doi: 10.1128/MCB.05036-11

Fig. 7.

Fig. 7.

KPNA6 modulates the antioxidant response by promoting ubiquitination and degradation of Nrf2. (A) KPNA6 is required for efficient repression of nuclear Nrf2 protein levels during the postinduction phase of the antioxidant response. MDA-MB-231 cells were transfected with either scrambled control siRNA or siRNA against KPNA6. At 48 h posttransfection, cells were either left untreated (mock) or treated with 100 μM tBHQ for 3 h (3). After removal of tBHQ by washing, cells were further incubated in normal medium for 3 h (3+P3, where “P” stands for postinduction) or for 6 h (3+P6). Nuclear extracts of the cells were analyzed by immunoblotting with anti-Nrf2, anti-KPNA6, and anti-lamin A antibodies. (B) KPNA6 represses the transcription of Nrf2 downstream target genes through posttranscriptional regulation of Nrf2. MDA-MB-231 cells were transfected with either control siRNA (■) or siRNA against KPNA6 (□). Cells were either untreated or treated with 50 μM tBHQ for either 16 h (upper and middle panels) or the indicated time period (lower panel) before being harvested and used for qRT-PCR analysis. Error bars indicate standard deviations of the results from three independent experiments. Asterisks indicate significant differences between two samples. (C) KPNA6 promotes polyubiquitination of Nrf2. MDA-MB-231 cells were transfected with siRNA as described above. At 48 h after transfection, cells were cotreated with 100 μM tBHQ and 10 μM MG132 for 4 h and harvested in denaturing conditions. Cell lysates were diluted and subjected to immunoprecipitation with an anti-Nrf2 antibody, followed by immunoblotting with an antiubiquitin antibody (α-Ub). (D) Half-life of Nrf2 protein is extended in the absence of KPNA6. MDA-MB-231 cells were transfected with siRNA as described above. At 48 h after transfection, cells were treated with 100 μM tBHQ for 4 h. Cells were then washed once with PBS and incubated with fresh medium containing 50 μM cycloheximide for the indicated times. Cell lysates were analyzed by immunoblotting with an anti-Nrf2 antibody. The relative intensities of the Nrf2 bands were quantified and plotted on a semilog scale. The calculated half-life of Nrf2 in each group is shown.