Hybond polyvinylidene difluoride membrane

Samples were made up in dissociation buffer (1× dissociation buffer (100 mm Tris-HCl, 2% (w/v) SDS, 10% (v/v) glycerol, 100 mm DTT, 0.02% (w/v) bromphenol blue, pH 6.8) and heated at 95 °C for 5 min. Proteins were resolved by SDS-PAGE on 7–17% acrylamide Tris-glycine gels and then transferred to Hybond polyvinylidene difluoride membranes (GE Healthcare). Following electrotransfer, the membranes were blocked for 1 h in PBS with 0.1% Tween 20 (PBST) and 5% (w/v) nonfat milk and then incubated with primary antibody overnight at 4 °C. Antigens were probed using the following primary antibodies: anti-APP (22C11, Millipore), SAF32 (anti-PrP N terminus, Cayman Chemical), 8H4 (anti-PrP residues 175–185), anti-ADAM10 antibody (Abcam), 6E10 (anti-Aβ(1–17), Merck Biosciences), AC15 (anti-β-actin) and synapsin 1 (Sigma), 2B3 (anti-human sAPPα, Immuno-Biological Laboratories), and anti-phospho-Src family kinase (Tyr-416; Cell Signaling Technology). Primary antibodies were detected by incubation with horseradish peroxidase–conjugated secondary antibody, both in PBST containing 2% BSA. Bound horseradish peroxidase conjugates were visualized using the ECL® detection system with a Syngene Gbox XT4 (Syngene). Densitometric analysis was performed using Genetools analysis software (Syngene).

Proteins extracted from cells were electrophoresed on 10% SDS-polyacrylamide gels and transferred to Hybond-polyvinylidene difluoride membranes (GE Healthcare UK Ltd., Amersham, UK). The membranes were incubated overnight at 4 °C with primary antibodies against AIF (1:1000, 5318; Cell Signaling Technology, Danvers, MA, USA), E1A (1:1000, 554155; BD Pharmingen, San Jose, CA, USA), p53 (1:1000, 18032; Cell Signaling Technology), and β-actin (1:5000, A-5441; Sigma-Aldrich, St. Louis, MO, USA), followed by incubation with secondary antibodies for 1 h at room temperature. The Amersham ECL chemiluminescence system (GE Healthcare UK Ltd.) was used to detect peroxidase activity of the bound antibody.