FIG. 4.

BBR induced Nrf2 activation in LPS-stimulated macrophages. Cells were treated as described in Fig. 2A. (A and B) Relative mRNA levels of NQO-1 and HO-1 were analyzed by qRT-PCR in RAW 264.7 cells (A) and in peritoneal macrophages (B). (C) In RAW264.7 cells transfected with pGFP-Nrf2 and pKeap1, subcellular localization of GFP-Nrf2 fusion protein in living cells was monitored under a fluorescent microscope after 24 h of transfection. (D) More than 100 GFP-positive cells were randomly selected and the percentage of cells with GFP in nucleus was expressed. (E) Cytoplasmic and nuclear fractions of RAW 264.7 cells were extracted and endogenious Nrf2 protein was probed with specific antibody via Western-blot assay. β-actin and H3 were probed as the controls for cytoplasm fraction and nucleus fraction, respectively. (F) Phosphorylated Nrf2 at Ser40 (p-Nrf2) and total Nrf2 in the whole cell lysates of RAW264.7 cells were probed by specific antibodies via Western-blot assay. Three separate experiments showed similar consequences, and one set of representative results are illustrated. *p<0.05 and **p<0.01, compared with control group in D. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars