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. 2020 Apr 29;71(5):1787–1801. doi: 10.1002/hep.31116

Figure 5.

Figure 5

P‐JNK regulates Nrf2 stability. (A) Activated JNK increases the polyubiquitination of endogenous Nrf2 in A549 cells. A549 cells were pretreated with 10 μM of MG132 for 3 hours, before 1‐hour treatment with 5 μg/mL of anisomysin in the continued presence or absence of 10 μM of MG132. Whole‐cell lysates were subjected to immunoprecipitation with anti‐Nrf2. Immunoprecipitates were analyzed by immunoblotting using antibody against Ub. Input, 10% of the cell lysate used for immunoprecipitation. The results presented are typical examples from at least three independent experiments. (B) Deletion of SDS1 in the Neh6 domain blocks degradation of Nrf2 induced by anisomycin. 293T‐mNrf2ΔE and 293T‐mNrf2ΔEΔSDS1 cells expressing Flag‐tagged mNrf2ΔETGE and Flag‐tagged mNrf2ΔETGEΔSDS1, respectively, were treated with 5 μg/mL of anisomysin for 15‐45 minutes. Whole‐cell lysates were immunoblotted with anti‐Flag or anti‐actin. Lower panel, relative levels of Flag‐mNrf2 mutants normalized to actin. The value at time 0 for the same protein was set at 1. Results are from three separate experiments. Values are mean ± SD. Abbreviations: IB, immunoblotting; Ub, ubiquitin.