Sodium dodecyl sulphate polyacrylamide gels

hNS1 cells cultivated in multiwell plates were dissociated with trypsin and centrifuged in PBS. The pellets were treated with lysis buffer (RIPA, Cell Signaling Technology, Danvers, MA, USA) to obtain cell extracts. Then, 50 μg of total proteins from cell extracts were loaded on sodium dodecyl sulphate-polyacrylamide gels (10%, BioRad, Hercules, CA, USA), electrophoresed (SDS-PAGE), and transferred to nitrocellulose membranes (GE Healthcare, Madrid, Spain). Membranes were blocked with milk (5%) and Tween20 (0.05%, Sigma) in TBS for 1 h and incubated overnight at 4 °C with mouse antibodies against APP (clone 22C11; 1:1000, Millipore, Merck, Darmstadt; Germany) and β-actin (1:1000, Sigma). Then, membranes were washed with Tween20 in TBS and incubated with HRP-conjugated antibody (horse anti-mouse peroxidase; 1:3000, Vector Laboratories, Newark, CA, USA) for 1 h. All antibodies used were diluted in milk and Tween20 in TBS. Visualization of immunoreactive bands was performed using ECL chemiluminescent system (Millipore) according to the manufacturer’s instruction. For quantification of blot images, densitometry analyses were realized using the software ImageJ v.1.54a. Band intensity was measured as a ratio of the protein of interest (APP using 22C11 antibody) to β-actin from three independent experiments.