MANF expression in the hippocampus.

<p><b>A</b>: Rat pups were sacrificed at the indicated postnatal days. MANF expression in the hippocampus was determined by IHC. CA1 and CA3, Cornu Ammonis 1 and 3 region of hippocampus; DG, dentate gyrus; SUB, subiculum. <b>B</b>: Image of higher magnification showed CA1, CA3 and DG of hippocampus. Scale bar = 100 µm.</p

MANF expression in the substantial nigra.

<p><b>A</b>: Rat pups were sacrificed at the indicated postnatal days. MANF IHC in substantial nigra was shown. SNC, Substantia Nigra pars Compacta; SNR, Substantia nigra pars reticulata. <b>B</b>: Double-labeling immunofluorescent staining was performed to determine the localization of MANF (green) in the SNC of PD15 pups. Dopaminergic neurons were indicated by tyrosine hydroxylase (TH) (red). The indicated square in the top panel is shown at a higher magnification in the bottom panel. TH- positive and negative cells were indicated by open arrows and solid arrows respectively. Scale bar = 100 µm.</p

Detection of MANF.

<p><b>A</b>: Different amount of human recombinant MANF (1–20 µg) were loaded on a SDS-polyacrylamide gel and subjected to immunoblotting analysis using a rabbit anti-MANF antibody. <b>B</b>: Rat pups of PD15 were sacrificed and perfused. The brain was dissected, sectioned and subjected to MANF immunohistochemistry (IHC) using a rabbit anti-MANF antibody (1∶6,000) as described in the Material and Methods. Rabbit serum (1∶6,000) was used as a control. Images were taken from the cerebral cortex. Scale bar = 100 µm.</p

MANF expression in the olfactory bulb.

<p><b>A</b>: Rat pups were sacrificed at the indicated postnatal days. MANF expression in the olfactory bulb was determined by IHC. 1, Glomerular layer (GLL); 2, External plexiform layer (EPL); 3, Mitral cell layer (MCL); 4, Internal plexiform layer (IPL); 5, Granule cell layer (GL). Scale bar = 100 µm. <b>B</b>: Images of higher magnification showed the MCL. Scale bar = 25 µm. <b>C</b>: Double-labeling immunofluorescent staining was performed to determine the identity of cells expressing MANF. The images showed the expression of MANF (green), NeuN (red), GFAP (red) and DAPI (blue) in the MCL of PD15 rat pups. Scale bar = 25 µm.</p

MANF expression in the hypothalamus.

<p>Rat pups were sacrificed at the indicated postnatal days. MANF expression in the hypothalamus was determined by IHC. IHC images were taken from the indicated area shown above. <b>A</b>: MANF expression in supraoptic nucleus (SON). <b>B</b>: MANF expression in tuberomammillary nucleus (TMN). OPT, optic tract. Scale bar = 100 µm.</p

MANF expression in the cerebellum.

<p><b>A</b>: MANF expression in the cerebellum of PD6 pups was determined by IHC. DN, deep cerebellar nuclei; LC, locus coeruleus. Scale bar = 100 µm. <b>B</b>: Rat pups were sacrificed at the indicated postnatal days. MANF expression in the cerebellum was determined by IHC. Scale bar = 100 µm. <b>C</b>: Images of higher magnification showing MANF expression in the external germinal layer (EGL), molecular layer (ML), Purkinje cells (PCs) and internal granule layer (IGL). Scale bar = 100 µm. <b>D</b>: Double-labeling immunofluorescent staining was performed to determine the identity of cells expressing MANF. The images showed the expression of MANF (green), NeuN (red), GFAP (red), Calbindin (red) and DAPI (blue) in the cerebellum of PD15 rat pups. Scale bar = 25 µm <b>E</b>: Co-localization of MANF (green) with tyrosine hydroxylase (TH) (red) in the LC of PD15 rat pups. Scale bar = 100 µm.</p

Study of developmental expression of MANF in the cerebral cortex by immunoblotting analysis.

<p><b>A</b>: Rat pups were sacrificed at the indicated postnatal days (PD1-30). The cerebral cortex was dissected and homogenized. Equal amount of sample was pooled (3–5 pups per group) and subjected to immunoblotting analysis using an anti-MANF antibody described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090433#pone-0090433-g001" target="_blank">Fig. 1</a>. <b>B</b>: The relative expression of MANF was quantified by densitometric analysis. & p<0.05, && P<0.01 compared with PD1; *p<0.05, **P<0.01, ***P<0.001 compared with PD3, and ### P<0.001, compared with other groups.</p

Effect of ethanol on p47^phox and p67^phox.

<p><b>A</b>. Top: SH-SY5Y cells were treated with ethanol (0 or 0.4%) for specified times. The expression of p47^phox and p67^phox was determined by immunoblotting. The expression of actin served as an internal loading control. Relative amounts of p47^phox and p67^phox was measured by densitometry and normalized to the expression of actin. Bottom: 7-days-old mice were exposed to ethanol as described under the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038075#s2" target="_blank">Materials and Methods</a>. The expression of p47^phox and p67^phox in the prefrontal cortex was determined with immunoblotting and quantified as described above. The experiment was replicated three times. The data are expressed as the mean ± SEM of three independent experiments. * denotes statistically significant difference from control group (<i>p</i><0.05). <b>B</b>. SH-SY5Y cells were exposed to ethanol (0 or 0.4%) for specified times. After the treatment, cells were lysed and the lysates were immunoprecipitated (IP) with an anti-p47^phox or anti-p67^phox antibody. The immunocomplexes were resolved by electrophoresis on a 10% SDS-polyacrylamide gel followed by immunoblotting analysis using either an anti-phosphoserine, anti-p47^phox, or anti-p67^phox antibody. <b>C</b>. Cytoplasm and membrane proteins were extracted as described under the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038075#s2" target="_blank">Materials and Methods</a>. The expression of p47^phox and p67^phox in the cytoplasm/membrane fractions was determined with immunoblotting. The expression of GAPDH and pan-cadherin served as internal loading controls for cytoplasmic and membrane fractions, respectively. Relative amounts of p47^phox and p67^phox was measured by densitometry and normalized to the expression of GAPDH or pan-cadherin. The experiment was replicated three times. The data are expressed as the mean ± SEM of three independent experiments. * denotes statistically significant difference from control group (<i>p</i><0.05).</p

Effect of p47^phox siRNA on ethanol-induced oxidative damage.

<p>SH-SY5Y cells were transfected with p47^phox siRNA for 48 hours and treated with ethanol (0 or 0.4%) for 48 hours. Protein oxidation and lipid peroxidation were determined by AOPP and MDA assay, respectively as described under the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038075#s2" target="_blank">Materials and Methods</a>. The data are expressed as the mean ± SEM of three independent experiments. * denotes statistically significant difference from control cells (<i>p</i><0.05); # denotes statistically significant difference from ethanol-treated cells (<i>p</i><0.05).</p

Effect of ethanol on NOX activation and ROS generation.

<p><b>A</b>. SH-SY5Y cells were exposed to ethanol (0 or 0.4%) for indicated times. NOX activity was determined by lucigenin chemiluminescence assay as described under the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038075#s2" target="_blank">Materials and Methods</a>. <b>B</b>. SH-SY5Y cells were pretreated with apocynin (0 or 50 μM) for 2 hours followed by exposure to 0.4% ethanol for 24 hours. Cells were labeled with DCFDA (10 μM) as described under the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038075#s2" target="_blank">Materials and Methods</a> and relative intensity of DCFDA (cellular ROS concentration) was measured and presented. The data are expressed as the mean ± SEM of three independent experiments. * denotes statistically significant difference from control cells (<i>p</i><0.05); # denotes statistically significant difference from ethanol-treated cells (<i>p</i><0.05).</p