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. 2013 Oct 9;33(41):16072–16087. doi: 10.1523/JNEUROSCI.1506-13.2013

Figure 3.

Figure 3.

Effect of cholesterol on amyloidogenic β- and γ-secretase cleavage. A, Effect of cholesterol on β-secretase BACE1. Dark-gray bars, Influence of cholesterol on β-secretase activity in purified membranes of human SH-SY5Y wt cells and ex vivo in purified membranes of mouse brains. Purified membranes were incubated with cholesterol (50nMol/mg protein) or solvent control. The β-secretase activity was determined by a fluorometric assay. Light-gray bars, Influence of cholesterol on gene expression and protein level of BACE1. SH-SY5Y wt cells were incubated with cholesterol (10 μm) or solvent control; total RNA was prepared and mRNA level of BACE1 was determined via RT-PCR. For the determination of BACE1 protein level, cell lysates were prepared, protein level adjusted, and equal volumes of cell lysates loaded and separated on 10–20% Tris-Tricine gels. WB analysis was performed with antibody B0806. A representative WB is shown. B, Influence of cholesterol on γ-secretase. Dark-gray bars, Influence of cholesterol on γ-secretase activity in purified membranes of human SH-SY5Y wt cells and ex vivo in purified membranes of mouse brains. Purified membranes were incubated with cholesterol (50nMol/mg protein) or solvent control and γ-secretase activity was determined by a fluorometric assay. Light-gray bars, Influence of cholesterol on gene expression of the γ-secretase components and on the protein level of the PS1 N-terminal fragment (PS1 NTF). SH-SY5Y wt cells were incubated with cholesterol (10 μm) or solvent control; total RNA was prepared and mRNA levels were determined via RT-PCR for PS1, PS2, APH1A, APH1B, PEN2, and NCSTN. For the analysis of the PS1 protein level, cell lysates were prepared, protein level adjusted, and equal volumes of cell lysates loaded and separated on 10–20% Tris-Tricine gels. WB analysis was performed with antibody sc7860. A representative WB is shown. A, B, Illustration and statistical significance as described for Figure 2; n ≥ 4. C, Determination of specificity of the β-secretase assay. The assay was performed as described using β-secretase inhibitor II (Calbiochem). D, Determination of specificity of the γ-secretase assay. The assay was performed as described using γ-secretase-inhibitor X (Calbiochem).