Figure 6. Zymosan-induced Nrf2 nuclear translocation is NADPH oxidase-dependent in isolated macrophages and in vivo.

Wild type (WT) and p47^phox−/− (CGD) bone marrow-derived macrophages (BMDMs) cultured in DMEM media with 10% serum were stimulated with zymosan (20 µ/ml). A) Western blot for Nrf2 and Tata box binding protein (TBP) in nuclear fractions of BMDMs from p47^phox−/− and WT mice at baseline, 1, 4, and 24 hours after zymosan. B) Densitometry from 3 separate experiments. p<.05 by ANOVA using Tukey post-test. C) Western blot showing expression of NQO1 in cytoplasmic extracts. D) Western blot for nuclear Nrf2 from gp91^phox−/ and p47^phox−/− macrophages treated with vehicle, zymosan (20 µg/ml), or the Nrf2 agonist electrophiles, CDDO-Im (1.0 µM) or sulforaphane (50 µM), for 4 hours. E) Nrf2 DNA binding activity of lung nuclear extracts in unstimulated (baseline) mice and 6 days after i.t. zymosan. n = 3−6 mice per group. Lung Nrf2 binding activity in zymosan-stimulated WT mice was significantly greater than that in unstimulated WT mice (student's t-test, p<0.05), whereas zymosan treatment had no effect on lung Nrf2 activation in CGD mice.