Western Blot Analysis of Sez6 Protein

The brain samples were heated at 95 °C in 1% SDS sample buffer with 100 mM DTT for 5 min, separated by SDS-PAGE on 4–20% precast polyacrylamide gels (Biorad, Feldkirchen, Germany), and transferred to PVDF membranes (Biorad, Feldkirchen, Germany). The membranes were blocked in 5% skim milk (Becton Dickinson, Heidelberg, Germany) in PBST (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) and incubated with anti-Sez6 antibody [24 (link)] or anti-Actin antibody (Merck Millipore, Darmstadt, Germany) at 4 °C overnight. The membranes were washed with PBST three times and incubated with HRP-conjugated secondary antibodies (Jackson Immunoresearch, West Grove, PA, USA) at room temperature for 1 h. After final washes with PBST, the membranes were incubated with ECL prime for a few minutes, and signal was detected with LAS-3000 (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). Densitometry quantification of Western blot signal was conducted using Multi Gauge V3.1 software (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan).