Journal: PLoS ONE

Article Title: Ferulic Acid Is a Nutraceutical β-Secretase Modulator That Improves Behavioral Impairment and Alzheimer-like Pathology in Transgenic Mice

doi: 10.1371/journal.pone.0055774

Figure Lengend Snippet: (A) Western blots are shown using an amino (N)-terminal APP polyclonal antibody (pAb N-APP; holo-APP is shown) and a carboxyl-terminal BACE1 polyclonal antibody (pAb BACE1). Western blots are also shown using an amino-terminal amyloid-β (Aβ) monoclonal antibody (mAb 82E1), which detects various amyloidogenic APP cleavage fragments including: Aβ monomer and oligomers as well as phospho-C99 (P-C99) and non-phospho-C99 (C99). Actin is shown as a loading control for each blot, and ratiometric densitometry data are shown below each lane. (B) Densitometry analyses are shown for ratios of C-99 or P-C99 to actin. (C) Aβ oligomers in the 2% SDS-soluble brain homogenate fraction were measured by sandwich ELISA. (D) Densitometry analysis is shown for ratio of BACE1 to actin. (E) α- and β-secretase activity assays are shown. Relative fluorescence units are depicted on the y-axis, and reaction time is represented on the x-axis. Representative Western blots for A were obtained from PSAPP mice treated with vehicle (PSAPP-V, n = 3) or with FA (PSAPP-FA, n = 3). Data for B-E were obtained from PSAPP mice treated with vehicle (PSAPP-V, n = 12) or with FA (PSAPP-FA, n = 12) for 6 months beginning at 6 months of age. All statistical comparisons are between PSAPP-V and PSAPP-FA mice.

Article Snippet: Briefly, BACE1 enzyme was incubated with various concentrations of FA (1.563, 3.125, 6.25, or 12.5 μM) or BACE1 inhibitor II (1.25 μM, as a positive control; Merck Millipore, Darmstadt, Germany) in the presence of 1×reaction buffer for 40 minutes prior to reading fluorescence values on a FLUOstar Omega (BMG LABTECH, San Diego, CA) fluorescent microplate reader.

Techniques: Western Blot, Control, Sandwich ELISA, Activity Assay, Fluorescence

Journal: PLoS ONE

Article Title: Ferulic Acid Is a Nutraceutical β-Secretase Modulator That Improves Behavioral Impairment and Alzheimer-like Pathology in Transgenic Mice

doi: 10.1371/journal.pone.0055774

Figure Lengend Snippet: (A) Amyloid-β (Aβ) 1–40 and Aβ 1–42 species were separately measured in cell supernatants from SweAPP N2a cells by sandwich ELISAs. (B) Inhibition of amyloidogenic APP processing in SweAPP N2a cells treated with various doses of FA. Western blots using an amino-terminal Aβ 1–17 monoclonal antibody (mAb 6E10), a carboxyl-terminal APP polyclonal antibody (pAb C-APP), or a carboxyl-terminal BACE1 polyclonal antibody (pAb BACE1) show holo-APP, carboxyl-terminal fragment generated by amyloidogenic APP cleavage (C99, β-CTF), and BACE1, respectively. Actin is included as an internal reference control, and ratiometric densitometry data are shown below each lane. (C) Densitometry results are shown as ratio of C99 to actin at various FA treatment doses. (D) Densitometry results are shown as ratio of BACE1 to actin at various FA treatment doses. (E) Quantitative real-time PCR results for BACE1 mRNA levels at various FA treatment doses are shown in arbitrary units, and BACE1 relative abundance is depicted on the y-axis. (F) Cell-free BACE1 activity assay results are displayed, and % of BACE1 activity is shown on the y-axis. (G) Lactate dehydrogenase (LDH) release assay results are shown for SweAPP N2a cells treated with 0 to 12.5 µM of FA. All statistical comparisons are vs. 0 µM of FA, and similar results were observed in 3–4 independent experiments.

Article Snippet: Briefly, BACE1 enzyme was incubated with various concentrations of FA (1.563, 3.125, 6.25, or 12.5 μM) or BACE1 inhibitor II (1.25 μM, as a positive control; Merck Millipore, Darmstadt, Germany) in the presence of 1×reaction buffer for 40 minutes prior to reading fluorescence values on a FLUOstar Omega (BMG LABTECH, San Diego, CA) fluorescent microplate reader.

Techniques: Inhibition, Western Blot, Generated, Control, Real-time Polymerase Chain Reaction, Activity Assay, Lactate Dehydrogenase Assay