Femto chemiluminiscent substrate

The proteins were transferred to a nitrocellulose membrane (66485, Pall Corporation, USA) using Trans Blot Turbo (1704270, Bio-Rad). Membranes were blocked in 10% non-fat milk in Tris buffered saline with Tween (TBST) at room temperature for 1 h and probed with primary antibodies diluted 1:1000-1:10 000 in 5% milk in TBST at 4 °C overnight. The primary antibodies were as follows: anti-drebrin (GTX12350, GeneTex; ab60933, Abcam), anti-GFP (GTX26673, Genetex; ab32146, Abcam), anti-AChR-α1, α3, α5 (838301, BioLegend), anti-rapsyn (ab156002, Abcam), anti-GAPDH (sc-25778, Santa Cruz) and anti-tubulin (ab18251, Abcam). After washing with TBST, the membranes were incubated with secondary antibodies conjugated to horseradish peroxidase [anti-rabbit HRP (111-035-144, Jackson Immuno Research, USA), anti-mouse HRP (7076, Cell Signaling, USA), anti-rat HRP (ADI-SAB-200-J, Enzo Life Sciences), and anti-goat HRP (sc-2020, Santa Cruz)]. Proteins were detected with Femto chemiluminiscent substrate (34095, ThermoFisher Scientific) and developed on X-ray films (771468, Carestream, USA).

Yeast cells were harvested and lysed with TCA 85% for 10 min at room temperature (RT). Supernatants were resolved in SDS-PAGE. Gels were transferred in polyvinylidene fluoride membranes (ImmobilonP, Millipore, Burlington, MA, USA), which were blocked for 1 h in tween-tris buffer saline (TTBS) plus 5% milk or 3% bovine serum albumin (BSA). Membranes were incubated o.n. at 4 °C with the following primary antibodies (Abs): 1:500 anti-GFP (Sigma, San Luis, MI, USA) and 1:500 anti-Aβ 6E10 (Covance, Princeton, NJ, USA). Membranes were washed thrice with TTBS and incubated for 1 h with 1:2000 anti-mouse secondary Abs (GE-Healthcare, Chicago, IL, USA). Three washes with TTBS were performed and membranes were developed with Super signal West Pico and Femto Chemiluminiscent substrate (Thermo Scientific, Waltham, MA, USA). Blotting quantification was performed with Quantity One software software (v.4.6.8).