Journal: The Journal of Biological Chemistry

Article Title: Combined treatment with the phenolics (−)-epigallocatechin-3-gallate and ferulic acid improves cognition and reduces Alzheimer-like pathology in mice

doi: 10.1074/jbc.RA118.004280

Figure Lengend Snippet: Combined therapy promotes nonamyloidogenic and inhibits amyloidogenic APP cleavage. Data were obtained from APP/PS1 mice that received vehicle (APP/PS1-V, n = 8), EGCG (APP/PS1-EGCG, n = 8), FA (APP/PS1-FA, n = 8), or EGCG plus FA (APP/PS1-EGCG/FA, n = 8) for 3 months starting at 12 months of age. Western blottings included each mouse (n = 8 per group), and quantitative data were averaged. Ten μg of protein from each sample was equally loaded per lane. Data for B–E are presented as standard deviations of the means. A, Western blots are shown using N-terminal APP polyclonal antibody (pAb N-APP), C-terminal anti-sAPP-β-sw mAb (mAb 6A1), and sAPP-α mAb (mAb 2B3). Western blots are also shown using N-terminal β-amyloid(1–17) (Aβ) mAb (mAb 82E1), which detects amyloidogenic APP cleavage fragments, including Aβ monomer and oligomers as well as phospho-C99 (P-C99) and nonphospho-C99 (C99). Actin is included as a loading control, and densitometry values are indicated below each lane. B–D, densitometry data are shown for ratios of sAPP-α or sAPP-β to APP as well as P-C99 or C-99 to actin. E, abundance of (N) 82E1 Aβ oligomers in the detergent-soluble brain homogenate fraction (measured by sandwich ELISA) are shown. Statistical comparisons for B–C and E are between-groups. Statistical comparisons for D are within each protein and between-groups. B, ***, p < 0.001 for APP/PS1-EGCG and APP/PS1-EGCG/FA mice versus APP/PS1-V and APP/PS1-FA mice; C–E, **, p < 0.01; ***, p < 0.001 for APP/PS1-V versus the other treated mice; ††, p < 0.01; †††, p < 0.001 for APP/PS1-EGCG/FA versus APP/PS1-EGCG or APP/PS1-FA mice. V, vehicle.

Article Snippet: Membranes were then hybridized for 1 h at ambient temperature with primary antibodies as follows: N-terminal anti-APP polyclonal (1:2,000 dilution, IBL); C-terminal anti-presenilin 1 (PS1) monoclonal (1:500 dilution, PS1-loop, Merck Millipore, Darmstadt, Germany); C-terminal anti-sAPP-α monoclonal (1:300 dilution, 2B3; IBL); C-terminal anti-sAPPβ-sw monoclonal (1:100 dilution, 6A1; IBL), N-terminal anti-Aβ(1–16) monoclonal (1:500 dilution, 82E1; IBL); C-terminal anti-BACE1 polyclonal (1:400 dilution, IBL); C-terminal anti-ADAM10 polyclonal (1:1,500 dilution, Cell Signaling Technology, Danvers, MA); anti-Cu/Zn SOD polyclonal (1:3,000 dilution, Enzo Life Sciences, Farmingdale, NY); anti-GPx1 polyclonal (1:4,000 dilution, Boster Biological Technology, Pleasanton, CA); and anti-β-actin monoclonal (1:5,000 dilution, 13E5; Cell Signaling Technology; as a loading control).

Techniques: Western Blot, Sandwich ELISA, Mouse Assay

Journal: The Journal of Biological Chemistry

Article Title: Combined treatment with the phenolics (−)-epigallocatechin-3-gallate and ferulic acid improves cognition and reduces Alzheimer-like pathology in mice

doi: 10.1074/jbc.RA118.004280

Figure Lengend Snippet: EGCG plus FA increases ADAM10 and decreases BACE1 expression. Data were obtained from APP/PS1 mice that were given vehicle (APP/PS1-V, n = 8), EGCG (APP/PS1-EGCG, n = 8), FA (APP/PS1-FA, n = 8), or EGCG plus FA (APP/PS1-EGCG/FA, n = 8) for 3 months starting at 12 months of age. Western blottings included each mouse (n = 8 per group), and quantitative data were averaged. Ten μg of protein from each sample was equally loaded per lane. Data for B–D are presented as standard deviations of the means. A, Western blots are shown using C-terminal ADAM10 (α-secretase candidate) polyclonal antibody (pAb ADAM10) and C-terminal BACE1 (β-secretase) polyclonal antibody (pAb BACE1). Actin is included as an internal loading control, and densitometry data are shown below each lane. Densitometry results are shown for ratios of precursor ADAM10 (pADAM10, B) or mature ADAM10 (mADAM10, C) to actin. D, densitometry data are shown for ratios of BACE1 to actin. Statistical comparisons for B–D are between-groups. B and C, ***, p < 0.001 for APP/PS1-EGCG and APP/PS1-EGCG/FA versus APP/PS1-V and APP/PS1-FA mice; D, ***, p < 0.001 for APP/PS1-V versus the other treated mice; †††, p < 0.001 for APP/PS1-EGCG/FA versus APP/PS1-EGCG or APP/PS1-FA mice. V, vehicle.

Article Snippet: Membranes were then hybridized for 1 h at ambient temperature with primary antibodies as follows: N-terminal anti-APP polyclonal (1:2,000 dilution, IBL); C-terminal anti-presenilin 1 (PS1) monoclonal (1:500 dilution, PS1-loop, Merck Millipore, Darmstadt, Germany); C-terminal anti-sAPP-α monoclonal (1:300 dilution, 2B3; IBL); C-terminal anti-sAPPβ-sw monoclonal (1:100 dilution, 6A1; IBL), N-terminal anti-Aβ(1–16) monoclonal (1:500 dilution, 82E1; IBL); C-terminal anti-BACE1 polyclonal (1:400 dilution, IBL); C-terminal anti-ADAM10 polyclonal (1:1,500 dilution, Cell Signaling Technology, Danvers, MA); anti-Cu/Zn SOD polyclonal (1:3,000 dilution, Enzo Life Sciences, Farmingdale, NY); anti-GPx1 polyclonal (1:4,000 dilution, Boster Biological Technology, Pleasanton, CA); and anti-β-actin monoclonal (1:5,000 dilution, 13E5; Cell Signaling Technology; as a loading control).

Techniques: Expressing, Western Blot, Mouse Assay

Journal: The Journal of Biological Chemistry

Article Title: Combined treatment with the phenolics (−)-epigallocatechin-3-gallate and ferulic acid improves cognition and reduces Alzheimer-like pathology in mice

doi: 10.1074/jbc.RA118.004280

Figure Lengend Snippet: Combination therapy with EGCG plus FA dampens neuroinflammation and oxidative stress. Data were obtained from APP/PS1 mice that received vehicle (APP/PS1-V, n = 8), EGCG (APP/PS1-EGCG, n = 8), FA (APP/PS1-FA, n = 8), or EGCG plus FA (APP/PS1-EGCG/FA, n = 8) for 3 months commencing at 12 months of age (mouse age at sacrifice = 15 months). Data for A and B additionally included WT mice treated in parallel with vehicle (WT-V, n = 8), EGCG (WT-EGCG, n = 8), FA (WT-FA, n = 8), or EGCG plus FA (WT-EGCG/FA, n = 8). Data for A and B as well as D and E are presented as standard deviations of the means. QPCRs are shown for tumor necrosis factor-α (TNF-α) or interleukin-1β (IL-1β) proinflammatory cytokines or for key oxidative stress markers superoxide dismutase 1 (SOD1) or GSH peroxidase 1 (GPx1). Data for A and B are expressed as relative fold over WT-V mice. C, Western blots are shown using anti-Cu/Zn SOD polyclonal antibody (pAb SOD1) or by anti-GPx1 polyclonal antibody (pAb GPx1). Western blots included each mouse (n = 8 per group), and quantitative data were averaged. Ten μg of protein from each sample was equally loaded per lane. Actin is included as a loading control for each appropriate blot, and densitometry data are shown below each lane. Densitometry data are shown for ratios of SOD1 to actin (D) or for GPx1 to actin (E). Statistical comparisons for A and B are between-groups but within each mRNA species. Statistical comparisons for D and E are within each protein but between-groups. *, p < 0.05; **, p < 0.01; ***, p < 0.001 for APP/PS1-V versus the other treated mice; †, p < 0.05; †††, p < 0.001 for APP/PS1-EGCG/FA versus APP/PS1-EGCG or APP/PS1-FA mice. V, vehicle.

Article Snippet: Membranes were then hybridized for 1 h at ambient temperature with primary antibodies as follows: N-terminal anti-APP polyclonal (1:2,000 dilution, IBL); C-terminal anti-presenilin 1 (PS1) monoclonal (1:500 dilution, PS1-loop, Merck Millipore, Darmstadt, Germany); C-terminal anti-sAPP-α monoclonal (1:300 dilution, 2B3; IBL); C-terminal anti-sAPPβ-sw monoclonal (1:100 dilution, 6A1; IBL), N-terminal anti-Aβ(1–16) monoclonal (1:500 dilution, 82E1; IBL); C-terminal anti-BACE1 polyclonal (1:400 dilution, IBL); C-terminal anti-ADAM10 polyclonal (1:1,500 dilution, Cell Signaling Technology, Danvers, MA); anti-Cu/Zn SOD polyclonal (1:3,000 dilution, Enzo Life Sciences, Farmingdale, NY); anti-GPx1 polyclonal (1:4,000 dilution, Boster Biological Technology, Pleasanton, CA); and anti-β-actin monoclonal (1:5,000 dilution, 13E5; Cell Signaling Technology; as a loading control).

Techniques: Western Blot, Mouse Assay

Journal: Biochimica et biophysica acta

Article Title: Pharmacological inhibition of NOX4 ameliorates alcohol-induced liver injury in mice through improving oxidative stress and mitochondrial function

doi: 10.1016/j.bbagen.2016.09.009

Figure Lengend Snippet: Protein levels of NOX4, alcohol oxidation enzymes, antioxidant enzymes and serum H2O2 in the liver of mice fed control (Ctrl) or ethanol (EtOH) for four weeks with or without GKT137831 for the last two weeks. (A) The immunoblot bands of NOX4 in the liver. (B) The immunoblot bands of NOX4 in the mitochondria. (C) The immunoblot bands of ADH, CYP2E1, Catalase, and ALDH2 in the liver. (D) The immunoblot bands of GSTM1, SOD1, SOD2, and Gpx1 in the liver. (E) The levels of H2O2 in the serum. Serum H2O2 was measured by Amplex Red Peroxide/Peroxidase Assay kit. The bands density was quantified by densitometry analysis. The ratio to HSP60 or β-actin was calculated by setting the value of ctrl as one. Results are mean ± SD (n=3). Results for bars that do not share a letter differed significantly among groups (P <0.05). Significant differences among groups are determined by ANOVA followed by Tukey’s test.

Article Snippet: The membrane was probed with polyclonal antibodies against NADPH oxidase 4 (NOX4, Cat. # ab133303), CYP2E1, aldehyde dehydrogenase 2 (ALDH2), oxphos (Abcam, Cambridge, MA), 4HNE (Northwest Life Science Specialties, Vancouver, WA), superoxide dismutase 1 (SOD1), glutathione S-transferase Mu 1 (GSTM1), Bax (Santa Cruz Biotechnologies, Santa Cruz, CA), calnexin, anti-heat shock protein 60 (HSP60) (BD, Franklin Lake, NJ), alcohol dehydrogenase (ADH), peroxisome proliferator-activated receptor alpha (PPAR-α), hepatocyte nuclear factor 4 (HNF-4α), glutathione peroxidase1 (Gpx1), (Novus biological, Littleton, CO), catalase, superoxide dismutase 2 (SOD2), voltage-dependent anion channel (VDAC) (Millipore, Billerica, MA), caspase-9, cleaved caspase-9, cleaved-PARP (Cell Signaling Technology, Danvers, MA) ACADVL, CPT-1 (Proteintech, Chicago, IL), and β-actin (Sigma-Aldrich) respectively.

Techniques: Western Blot

Journal: Biochimica et biophysica acta

Article Title: Pharmacological inhibition of NOX4 ameliorates alcohol-induced liver injury in mice through improving oxidative stress and mitochondrial function

doi: 10.1016/j.bbagen.2016.09.009

Figure Lengend Snippet: The protein levels of mitochondrial 4HNE and Gpx1, mitochondrial respiratory complexes, the number of mtDNA, and the concentration of ATP in the liver of mice fed control (Ctrl) or ethanol (EtOH) for four weeks with or without GKT137831 for the last two weeks. (A) The immunoblot bands of mitochondrial 4HNE and Gpx1. (B) The immunoblot bands of mitochondrial respiratory complex I-V. (C) The mtDNA levels were measured with NADH dehydrogenase subunit 6 by qPCR. (D) ATP levels in the liver of mice were detected by commercial ATP assay kit. The bands density was quantified by densitometry analysis. The ratio to β-actin or mitochondrial HSP60 was calculated by setting the value of ctrl as one. Results are mean ± SD (n=3). Results for bars that do not share a letter differed significantly among groups (P <0.05). Significant differences among groups are determined by ANOVA followed by Tukey’s test.

Article Snippet: The membrane was probed with polyclonal antibodies against NADPH oxidase 4 (NOX4, Cat. # ab133303), CYP2E1, aldehyde dehydrogenase 2 (ALDH2), oxphos (Abcam, Cambridge, MA), 4HNE (Northwest Life Science Specialties, Vancouver, WA), superoxide dismutase 1 (SOD1), glutathione S-transferase Mu 1 (GSTM1), Bax (Santa Cruz Biotechnologies, Santa Cruz, CA), calnexin, anti-heat shock protein 60 (HSP60) (BD, Franklin Lake, NJ), alcohol dehydrogenase (ADH), peroxisome proliferator-activated receptor alpha (PPAR-α), hepatocyte nuclear factor 4 (HNF-4α), glutathione peroxidase1 (Gpx1), (Novus biological, Littleton, CO), catalase, superoxide dismutase 2 (SOD2), voltage-dependent anion channel (VDAC) (Millipore, Billerica, MA), caspase-9, cleaved caspase-9, cleaved-PARP (Cell Signaling Technology, Danvers, MA) ACADVL, CPT-1 (Proteintech, Chicago, IL), and β-actin (Sigma-Aldrich) respectively.

Techniques: Concentration Assay, Western Blot, ATP Assay