Journal: The American Journal of Pathology

Article Title: Pigment Epithelium-Derived Factor Is an Intrinsic Antifibrosis Factor Targeting Hepatic Stellate Cells

doi: 10.2353/ajpath.2010.091085

Figure Lengend Snippet: A and B: PEDF prevents JunD and NF-κB nuclear translocation in a PPARγ-dependent manner. A: Primary rat HSCs were assayed by dual immunofluorescence staining for PPARγ (red) and JunD (green). Cells were pretreated with 10 μmol/L PPARγ antagonist (GW9662) for 1 hour and then with or without PEDF for an additional 48 hours. Nuclei were stained with 4′,6-diamidino-2-phenylindinole. B: The inhibitory effect of PEDF on the nuclear translocation of JunD and NF-κB in HSC-T6 cells is blocked by PPARγ siRNA pretreatment. Nuclear and cytoplasmic fractions of HSC-T6 cells were prepared and subjected to immunoblot analysis with antibodies as indicated. Antibodies against histone H1 were used to validate the fractionation procedure. C and D: PPARγ associates with JunD and p65/IκBα. HSC-T6 cells were pretreated with 20 μmol/L PPARγ antagonist (GW9662 or G3335) or DMSO solvent as indicated for 1 hour and then with or without PEDF for an additional 24 hours before the preparation of cell lysates. Immunoblotting with various antibodies as indicated was performed on respective anti-PPARγ (C) or anti-p65 (D) immunoprecipitates. Lysates were also analyzed for total JunD, IκBα, and β-actin before immunoprecipitation (input).

Article Snippet: Pre-cleared lysates were incubated with anti-PPARγ antibody (20 μg; sc-7273; Santa Cruz Biotechnology) or NF-κB p65 (5 μg; #3034; Cell Signaling Technology), and Protein G Sepharose at 4°C for 4 hours.

Techniques: Translocation Assay, Immunofluorescence, Staining, Western Blot, Fractionation, Immunoprecipitation

Journal: PLoS ONE

Article Title: Cdc42-Dependent Activation of NADPH Oxidase Is Involved in Ethanol-Induced Neuronal Oxidative Stress

doi: 10.1371/journal.pone.0038075

Figure Lengend Snippet: A : 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 (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 ( p <0.05). 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 (bottom panel). The data are expressed as the mean ± SEM of three independent experiments. * denotes statistically significant difference from control cells ( p <0.05); # denotes statistically significant difference from ethanol-treated cells ( p <0.05). C . 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 . D . 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 . 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 ( p <0.05). # denotes statistically significant difference from ethanol-treated cells ( p <0.05).

Article Snippet: Briefly, for immunoprecipitation, an aliquot of cell lysates containing 200 µg of proteins were pre-cleared with protein G-Sepharose beads (Amersham Biosciences, USA).

Techniques: Western Blot, Expressing, Transfection, Dominant Negative Mutation, Activity Assay