Western Blot Analysis of Amyloid-Beta and GAPDH

Protein lysates in 1X Laemmli buffer (Sigma-Aldrich) were separated on an SDS-10% polyacrylamide gel (Sigma-Aldrich) and the proteins were transferred onto nitrocellulose membranes (GE Healthcare, Buckinghamshire, UK). The membranes were blocked in TBS (Fisher Scientific, Loughborough, UK) containing 5% dried milk powder (w/v) and 0.1% Tween-20 (Fisher Scientific), for 1 h at room temperature. Following 3 washes with TBS-0.1% Tween-20, the nitrocellulose membranes were incubated with primary antibodies against Aβ⁴² and GAPDH (both from Cell Signalling Technology, Danvers, MA, USA). The primary antisera were used at a 1:1,000 dilution overnight at 4°C. The membranes were washed thoroughly for 30 min with TBS-0.1% Tween-20 prior to incubation with the secondary antibody, HRP-conjugated immunoglobulin (1:2,000; Sigma-Aldrich), for 1 h at room temperature and further washing for 30 min with TBS-0.1% Tween-20. Antibody complexes were visualised as previously described (21 (link)).