Synaptosomal Isolation and Protein Analysis

Synaptosomal isolation and western blots were performed as previously described (Naviaux et al. 2013 (link); Naviaux et al. 2015 (link)). Briefly, cerebral samples were collected, homogenized, and synaptosomes isolated by discontinuous Percoll gradient centrifugation. Twelve μg of cerebral synaptosomal protein was loaded. Blots were probed with primary antibodies overnight in a cold room using anti-mGluR1 (#ab27199 from Abcam, Cambridge, MA), mGluR5 (#ab76316 from Abcam), and dopamine D₄ receptor (#ADR-004, from Alomone Labs, Jerusalem, Israel) antibodies. After washing, the membranes were blotted with goat anti-rabbit secondary antibody (#31460 from Pierce, Rockford, IL). The proteins of interest were visualized by ECL reagent (#32109) or Pierce SuperSignal^™ West Femto Maximum Sensitivity Substrate (#PI-34095) and the immunoblots were exposed to X-Omat Blue films and scanned. The target protein density was normalized by Ponceau S staining and analyzed in GraphPad Prism 6.0 (La Jolla, CA).