FIG. 3.

TAK1 does not inhibit Nrf2 degradation machinery. (A) HEK293 cells were transfected with expression vectors for Nrf2, Keap1, TAK1, and TAB1 as indicated, and at 48 h post-transfection, 100 μg/ml cycloheximide (CHX) was added to the culture medium. Cells were harvested at 0, 0.5, 1.0, and 1.5 h after CHX addition (lanes, 1–4). Cell lysates were analyzed by immunoblotting. A stable protein, β-actin, is used as a loading control. (B, C) The immunoblotting band intensity of Nrf2 was quantified by ImageJ software and normalized to the intensity of the β-actin band. Three independent experiments were performed. Means ± SEM are shown (B). Nrf2 protein half-lives were calculated by GraphPad software using an exponential decay equation model. The bars depict the 95% confidence intervals (C). NS, not significant (two-tailed unpaired Student's t-test).