Pre casted gels

Western blotting was used to measure APP levels in brain extractions. The proteins were extracted from the cortex using PBS or RIPA buffer (Sigma-Aldrich, Germany) with proteinase and phosphatase inhibitors. The blots were run using pre-casted gels (4–20% from Bio-Rad) in TGS buffer (Bio-Rad). The proteins were transferred to nitrocellulose membranes (Bio-Rad) using the TransBlot turbo system from BioRad. The membranes were blocked for 1h with skim milk at 3% in PBS, then washed 3 × 10 mins in PBS and Tween-20 at 0,1%, after which they were incubated with primary and secondary antibodies diluted in PBS-Tween 20 at 0.1%. To develop the blot we use ECL Clarity (Bio-Rad) according to the manufacturer’s protocol and ChemiBlot XRS + system from Bio-Rad.

The preparation of whole cell lysates and western blot analysis of protein expression were performed according to the previous routine procedure [11 (link)]. In brief, total protein was extracted from H9C2 cells and protein concentration was determined using the Bicinchoninic Acid protein detection kit (Sigma-Aldrich). The whole cell lysis buffer was mixed with SDS loading buffer. After centrifugation at 13,000 g at 4 °C for 20 min, the supernatants were collected. Equal amounts of proteins (50 μg) were subjected to SDS-PAGE on 10 or 5% pre-casted gels (Bio-Rad, Hercules, CA, USA). The supernatant was collected after centrifugation at 13,000 g at 4 °C for 20 min. According to the previously established protocol, protein expression was detected [11 (link)]. The primary antibodies used were as follows: anti-SORBS2 (1:200, SAB4200183, Sigma-Aldrich), anti-ENH (1:200, SAB2101761, Sigma-Aldrich) and anti-actin (1:500, A5385, Sigma-Aldrich). The secondary antibody used were anti-mouse IgG (1:10000, A5385, Sigma-Aldrich), anti-rabbit IgG (1:10000, B7389, Sigma-Aldrich). Quantification was performed by measurement of the intensity of the signals with aid of ImageJ (NIH, Bethesda, MD, USA).