Hybond p

Cells were lysed with RIPA buffer in the presence of proteases and phosphatase inhibitor mixture. Forty microgram of total proteins was separated by 8–10% Tris Glycine SDS-PAGE (Invitrogen) and transferred to a polyvinylidene difluoride membrane (Hybond-P, Amersham, Buckinghamshire, UK). Membranes were blocked in Tris-buffered saline with 2% BSA and 0.2% Tween 20 and incubated overnight at 4 °C with primary antibodies listed in Supplementary Table S1. Immunoreactive bands were detected with peroxidase-conjugated secondary antibodies listed in Supplementary Table S2 and with ECL Plus detection system (Amersham).

Presence and relative levels of the amyloid-β peptides and APOE protein in each SDG fraction were assessed by dot-blot analysis on nitrocellulose membrane (Hybond P, Invitrogen), when the experiment was run on purified proteins in PBS. The blots were prepared by applying 2 µl of each fraction on the membrane. All fractions were applied on the same blot. The optical density of each dot was calculated by the ImageQuant™ TL software (v5, Invitrogen).
APOE protein was detected by the A1.4 antibody (1/8000, sc-13521, Santa Cruz Biotech.) and the secondary HRP-conjugated anti-mouse antibody (1/1000, sc-2005, Santa Cruz Biotech.) in
^5%M-TBS-T
^0.3%blocking solution (10 mM TBS containing 5% non-fat dry milk, 170-6404, Bio-Rad and 0.3% Tween-20). Amyloid-β peptides in the fractions were detected by the 6E10 mouse monoclonal antibody (1/1000, SIG-39320, Covance) and the secondary HRP-conjugated antibody (sc-2005, Santa Cruz Biotech.).