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. 2023 Aug 9;12(8):1586. doi: 10.3390/antiox12081586

Figure 3.

Figure 3

Mutation of the AMPK-dependent phospho-sites to alanine on Nrf2 impedes its βTrcP2-mediated ubiquitination and degradation in Keap1−/− cells. (A–E) Keap1-null MEFs were transfected with the indicated expression plasmids for 48 h. (A) Ubiquitination assay of EGFP-Myc tagged Nrf2 expression plasmids in Keap1-null MEFs, after co-transfection with His-tagged Ubiquitin and the indicated HA-βTrCP2 construct. (B–E) Expression levels of EGFP-Myc tagged WT-Nrf2 (B,C) or EGFP-Myc tagged TM-Nrf2 (D–E) with or without co-transfection of HA-tagged βΤrCP2 in the presence or absence of the AMPK activator A 769662 (50 μM, 4 h). Cell lysates were subjected to immunoblot analysis for MYC, HA, or actin. Representative blots (B,D) and quantification analysis (C,E) of 3 independent experiments are depicted. Plus symbols in the tables indicate the different constructs/chemicals used in the various experimental conditions. Data are presented as means ± SEM. Statistical analysis was performed using multiple comparison one-way ANOVA, with Sidak’s correction, * p < 0.05 and ** p < 0.01. ns: not significant.