Biorad wet transfer system

Frozen brain samples were transferred into ice-cold radioimmunoprecipitation assay (RIPA) buffer (Santa Cruz) – containing protease inhibitors, phenylmethylsulfonyl fluoride, and sodium orthovanadate –, homogenized with a polytron, and spun down twice for 20 min at 13000 rpm at 4 °C to remove cell debris. Protein concentration was quantified using a bicinchoninic acid assay (Pierce BCA protein assay kit, Thermo Fisher Scientific) according to the manufacturer’s instruction. Equal amounts of protein (20 μg per lane) were loaded, and electrophoresis was performed in NuPAGE 4 to 12% gradient Bis-Tris polyacrylamide protein gels (Thermo Fisher Scientific). Transfer was performed using PVDF membranes with the Biorad wet transfer system (Bio-Rad Laboratories). Membranes were blocked with 5% milk in PBS with 0.1% Tween-20 (Sigma Aldrich), incubated overnight with primary antibodies (Table S3) at 4°C, washed, and incubated with secondary antibody for 2 h at room temperature. After a final wash, secondary antibodies were visualized using Pierce ECL chemiluminescence reagents (Thermo Fisher Scientific) and the signal was captured with X-ray films (Thomas Scientific).