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. Author manuscript; available in PMC: 2016 Dec 1.
Published in final edited form as: Free Radic Biol Med. 2015 Nov 5;89:1184–1191. doi: 10.1016/j.freeradbiomed.2015.11.003

Figure 4.

Figure 4

Both Nrf2 synthesis and degradation decline with age.

(A) Hepatocytes from young and old rats were treated with 50 μM A3T, 100 μM lipoic acid (LA), and/or 100 nM bortezomib for 6 h. Nuclear fractions were isolated from the harvested samples and assayed for Nrf2 content. Samples were quantitated by densitometry relative to the loading control Actin. Statistical analysis was done by two-way ANOVA and Tukey's post-hoc method. *p<0.05 vs young control without BTZ. (N=3). (B) Graphic representation of Keap1 immunoblots using liver tissue from young and old rats N=4; *p <0.05. Actin loading control is from the same western blot membrane shown in Figure 1A. (C) Half-life of Nrf2 protein was determined by transfecting primary hepatocytes from young and old rats with the pHA-Nrf2 expression vector, then adding 100 μM cycloheximide 20 h post-transfection. Samples were taken every 20 minutes and protein was extracted. Nrf2 protein levels were determined by immunoblot, quantified by ImageJ, and used to create the graph. N=3.