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 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

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 p38 MAPK inhibitor on PIC-stimulated cell signaling.

<p>MDA-MB231 cells were pretreated with a selective p38 MAPK inhibitor (SB203580, 5 µM) for 2 hours followed by PIC (0 or 10 ng/ml) treatments for an additional 6 hours. <b>A</b>: After the treatment, cell lysates were collected for immunoblotting analysis of the phosphorylation/expression of PKR, p38 MAPK, MK2, LIMK1 and cofilin. The expression of actin served as a loading control. <b>B</b>: The relative levels of pPKR, pp38, pMK2, pLIMK and pcofilin were quantified as described under the Materials and Methods and normalized to the expression of PKR, p38 MAPK, MK2, LIMK1 and cofilin respectively. * denotes a significant difference from untreated controls. <b>C</b>: The expression of phospho-cofilin and phospho-MK2 was visualized by immunofluorescent staining as described under the Materials and Methods. Scale bar  = 100 µm. <b>D</b>: MDA-MB231 cells were pretreated with SB203580 (5 µM) for 2 hours then placed into the upper compartments of migration chambers (transwells) in the presence of PIC (0 or 10 ng/ml). The number of MDA-MB231 cells that migrated through the transwells was determined as described under the Materials and Methods. The experiment was replicated three times. * denotes a statistically significant difference from controls. # denotes a significant difference from PIC-treated 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

Role of Cdc42 in ethanol-induced NOX activation and ROS generation.

<p><b>A</b>: SH-SY5Y cells were exposed to ethanol (0 or 0.4%) for indicated times. Active Cdc42 was pulled down by PAK1-PBD color agarose beads and analyzed by immunoblotting using an anti-Cdc42 antibody 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> (top panel). Relative expression of active Cdc42 was quantified (bottom panel). The data are expressed as the mean ± SEM of three independent experiments. * denotes statistically significant difference from control cells (<i>p</i><0.05). <b>B</b>. SH-SY5Y cells were transfected with dominant-negative Cdc42 (DN-Cdc42) for 48 hours to inhibit endogenous Cdc42 activity. Active Cdc42 was determined as described above (top panel). After transfection with DN-Cdc42, cells were exposed to ethanol (0.4%, 24 hours). The effect of ethanol on NOX activity and ROS generation was investigated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038075#pone-0038075-g001" target="_blank">Figure 1</a> (bottom panel). 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). <b>C</b>. SH-SY5Y cells were transfected with DN-Cdc42 for 48 hours. After transfection, cells were exposed to ethanol (0.4%, 24 hours). The effect of ethanol on p47^phox phosphorylation was investigated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038075#pone-0038075-g002" target="_blank">Figure 2B</a>. <b>D</b>. SH-SY5Y cells were transfected with DN-Cdc42 for 48 hours. After transfection with DN-Cdc42, cells were exposed to ethanol (0.4%, 24 hours). 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 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 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 cells (<i>p</i><0.05). # denotes statistically significant difference from ethanol-treated cells (<i>p</i><0.05).</p