Skip to main content
. 2019 Sep 6;8:e42951. doi: 10.7554/eLife.42951

Figure 5. Suppression of Nrf2 protein levels by Rgs12 is dependent on the proteasome degradation pathway.

(A) Diagram summarizing the inhibitors of the Keap1-proteasome axis to modulate Nrf2 protein levels. (B) RAW264.7 cells stably-transfected with Rgs12-His or empty vector treated with increasing doses of tBHQ. (C) Nrf2 and Keap1 protein levels were quantified by densitometry analysis and normalized to β-actin (N = 3, *p<0.05, **p<0.01). (D) Western blot to detect Nrf2 and Keap1 in RAW264.7 cells stably-transfected with empty vector or Flag-Rgs12. RAW264.7 cells were treated with a combination of RANKL (100 ng/mL, 72 hr) and the proteasome inhibitor MG-132 (25 μM, 4 hr). (E) Nrf2 and Keap1 protein levels were quantified by densitometry analysis and normalized to β-actin (N = 3, *p<0.05, **p<0.01, ***p<0.001). (F) qPCR analysis of Nrf2 and Keap1 transcript levels in RAW264.7 cells transfected with Rgs12-His or empty vector. Data are means ± SD. Two-tailed t test was performed (N = 3, *p<0.05). tBHQ, tert-butylhydroquinone.

Figure 5.

Figure 5—figure supplement 1. Complete western blots shown in Figure 5.

Figure 5—figure supplement 1.

(A) Complete western blots used in Figure 5B. Sections shown in Figure 5B are highlighted with dashed boxes. Transfected RAW264.7 cells were induced with the indicated dosages of tBHQ for 4 hr. (B) Complete western blots used in Figure 5D. Sections shown in Figure 5D are highlighted with dashed boxes. RAW264.7 cells stably-transfected with empty vector or Flag-Rgs12 were treated with the following: RANKL (100 ng/mL, 72 hr), MG-132 (25 μM, 4 hr), and/or tBHQ (25 μM, 4 hr). tBHQ, tert-butylhydroquinone.