Effect of Metal Chelators on γ-Secretase Indicates That Calcium and Magnesium Ions Facilitate Cleavage of Alzheimer Amyloid Precursor Substrate
Figure 5
Effect of Ca2+ and Mg2+ on the size of γ-secretase complexes analyzed by BNPAGE and size-exclusion chromatography. 10 μg samples of protein extract from SHSY5Y membranes were incubated with HEPES buffer, pH 7.3, with or without additives, as indicated. EGTA and EDTA concentrations were 2 mM. CaCl2 and MgCl2 concentrations were 3 mM. (a) The incubations were mixed with BN-PAGE sample buffer and electrophoresed on 3–8% Tris-acetate gels. Proteins were transferred to PVDF membrane and the blot probed with anti-PS1 antibody 98/1. (b) SH-SY5Y membranes (200 μg per experiment) were extracted with 0.5% CHAPSO in buffer containing either 2 mM EDTA or 3 mM MgCl2. The samples were chromatographed on superose-6 size-exclusion column and eluted fractions were analysed by western blotting with PS1 N-terminal antibody 98/1. Western blot signals were quantified and plotted against fraction numbers.
