Mouse Brain Protein Expression Analysis

Mice brain were prepared as previously described [63 (link)]. Briefly, mice brain homogenized with RIPA buffer (Thermo Fisher Scientific, Rockford, IL, USA), and incubated on ice for 60 min, and centrifuged at 13,000 rpm for 20 min at 4 °C. An equal amount of total protein (30 μg) was subjected to SDS-PAGE (12%), and the membranes were incubated with the following primary antibodies: CB1 (1:500, Abcam, Cambridge, MA, USA), GAD67 (1:1000, Abcam, Cambridge, MA, USA), β-actin (1:1000, Abcam, Cambridge, MA, USA), and GAPDH (1:500, Cell Signaling Technology, Danvers, MA, USA). The membranes were then incubated with horseradish peroxidase-conjugated anti-rabbit and anti-mouse secondary antibodies (1:5000, Sigma-Aldrich, St. Louis, MO, USA). Immunoreactivity was visualized with an ECL Plus detection system (GE Healthcare, Chicago, IL, USA). The relative density of the protein bands was analyzed with ImageJ.