Nrf1b-MafG complex binds the ARE.

<p>(<b>A</b>) EMSA studies were performed with Nrf1b and MafG proteins generated by in vitro transcription and translation, and biotinylated DNA probe containing a consensus antioxidant response element described in Materials and Methods. Rabbit anti-Nrf1, anti-MafG, and IgG were used for super-shifts. Chevrons and arrows indicate shifted and super-shifted bands, respectively. (<b>B</b>) HEK293 cells transfected with Nrf1b-V5 were harvested 48 h after, and lysates were immunoprecipitated with anti-V5 antibody. Immunoprecipitates were then subjected to immunoblotting with anti-V5 or anti-MafG antibodies.</p

Nrf1b activates ARE-driven genes.

<p>Luciferase reporter bearing 3 copies of ARE (<b>A</b>)<b>,</b> or GCLM-luciferase reporter (<b>B</b>) was co-transfected with either Nrf1a or Nrf1b expression plasmid. Luciferase activities were normalized to <i>Renilla</i> luciferase from pRL-TK. Results are expressed relative to luciferase activities observed with vector alone. Histograms show the means of three separate experiments ± SD carried out in triplicates. *P<0.05 (<b>C</b>) Induction of endogenous ARE target genes. NIH3T3 cells were transfected with the indicated plasmids, and mRNA was harvested and analyzed 2 days after transfection by qRT-PCR. Histograms show mean ± SD (n = 4).</p

Nrf1b expression is widely distributed.

<p>Nrf1a and Nrf1b mRNA expression patterns were analyzed by RT-PCR in various cell lines (<b>A</b>) and mouse tissues (<b>B</b>)<b>.</b> Nrf1a and Nrf1b cDNA was amplified by PCR for 30 cycles and 18S was amplified for 20 cycles. Histograms show relative Nrf1a and Nrf1b expression normalized against 18S. (<b>C</b>)<b>.</b> Western blot of different mouse tissues probed with Nrf1 antibody. HEK293 cells transfected with pEF1-Nrf1a (lane 1), and pEF1-Nrf1b (lane 2) were used as controls for detection of the Nrf1a and Nrf1b isoforms by the Nrf1 antibody. Beta-actin was used as a loading control.</p

Nrf1b encodes a novel Nrf1 protein.

<p>Schematic diagram of human and mouse Nrf1 genomic sequences, and depiction of Nrf1a and Nrf1b transcripts. Solid and open boxes represent coding regions and untranslated regions, respectively. Solid lines represent introns and 5′-flanking regions.</p

Nrf1b is derived by alternative promoter usage.

<p>(<b>A</b>)<b>.</b> Nucleotide sequence and identification of putative cis-acting elements in the 5′-flanking region of Nrf1b exon 1. Numbering is relative to the first nucleotide of the initiation codon (ATG) designated as +1. Vertical arrow represents the transcription start site identified by primer extension analysis, and coding region is underlined. The cis-acting elements containing consensus sequences are boxed. (<b>B</b>)<b>.</b> Primer extension result with NIH3T3 mRNA. The peak corresponding to a 108-bp elongation product (FAM-labeled cDNA) is indicated by an arrow. Labeled cDNA was aligned with the sequence electropherogram to identify the base at which transcription starts for Nrf1b. Nucleotides are indicated on the x-axis. (<b>C</b>)<b>.</b> Relative luciferase activities of the mouse Nrf1b promoter construct in HEK293 and Hepa1c1c7 cells. Negative control consisted of the pGL3-Basic control. Luciferase activities were normalized to <i>Renilla</i> luciferase from pRL-TK. Histograms show mean ± SD of three independent experiments with triplicate samples per experiment. *P<0.05.</p

Nrf1b protein is localized in the nucleus.

<p>(<b>A</b>)<b>.</b> Epifluorescent micrographs showing COS7 cells transfected with Nrf1b-EGFP. (<b>B</b>) Quantitative analysis of the results in (B). The subcellular localization of EGFP and Nrf1b-EGFP was scored as follows: N>C, predominantly nuclear; N  =  C, evenly distributed between the nucleus and cytoplasm; NC)<b>.</b> Distribution of Nrf1b-V5 in cells. V5-tagged Nrf1b was harvested from HEK293 cells 48hr after transfection as described in the methods section, and analyzed by Western blot. The antibodies used for Western blotting are indicated on the right. Pyruvate kinase was used as a cytoplasmic marker, calnexin as an ER membrane marker, and lamina-associated polypeptide 2α (LAP2α) as a nuclear marker.</p

Immunohistochemical analysis of CD133, Fn14, TFF3, and HNF4α in regenerating livers of Nrf2+/+ and Nrf2−/− mice.

<p>Liver sections were prepared from the livers isolated at 60 h after PH from three mice per genotype. All liver sections were subjected to immunostaining with primary antibodies against CD133, Fn14, TFF3, or HNF4α. Representative immunohistochemically stained liver sections are shown.</p

Changes in hepatocyte density after partial hepatectomy (PH) in Nrf2+/+ and Nrf2−/− mice.

<p>Livers were collected from the two genotype groups of mice at the indicated time points post-PH. Liver sections were prepared and subjected to hematoxylin and eosin staining. Hepatocytes were counted in five randomly chosen fields per liver section (400x magnification) with Image-Pro Plus software. The results are shown as the means per field ± SD (n = 3 to 5 mice/time point/genotype; *, <i>p</i><0.05 between Nrf2+/+ and Nrf2−/− mice).</p

The mRNA expression of a group of genes associated with liver functions in regenerating livers of Nrf2+/+ and Nrf2−/− mice.

<p>Total RNA was prepared from the livers at the indicated time points after partial hepatectomy (PH). Hepatic expression levels of the genes indicated were measured by qRT-PCR and are expressed as the means of fold changes relative to the mRNA level in normal livers in Nrf2+/+ mice ± SD (n = 3 mice/time point/genotype; *, <i>p</i><0.05 between Nrf2+/+ and Nrf2−/− mice). NL, normal liver.</p

Protein expression of CD133, Fn14, TFF3, HNF4α, p-Akt1 (T308), Akt1, p-p70S6K (T389), p70S6K, p-4E-BP1 (T37/46), and 4E-BP1 in regenerating livers of Nrf2+/+ and Nrf2−/− mice.

<p>Livers were collected from normal mice and the mice subjected to partial hepatectomy (PH) at the indicated time points following surgery. Liver lysates prepared from three mice per time point per genotype were pooled with equal amount of protein from each preparation. Western blotting was performed with antibodies against the proteins indicated. Glyceraldehyde 3-phosphate dehydrogenase (GADPH) was used as a loading control. NL, normal liver.</p