Striatal Protein Analysis by SDS-PAGE and WB

SDS-PAGE and western blotting experiments were performed as previously described [40 (link)]. Striatal tissues were isolated and homogenized in lysis buffer (150 mM NaCl, 50 mM Tris, 1% Triton) supplemented with protease and phosphatase inhibitors. Proteins were separated using the NuPAGE system on 4–12% pre-cast gradient gels and MES/SDS running buffer (Thermo Fisher Scientific, Waltham, MA) and then were transferred to Immobilon P transfer membrane (Millipore, Burlington, MA) using a Mini Blot Module (Thermo Fisher Scientific, Waltham, MA). Blots were probed with anti-tyrosine hydroxylase (TH) antibodies (MAB318, Millipore, Burlington, MA) and anti-actin HRP-coupled antibodies (A3854, Sigma-Aldrich, St. Louis, MO). HRP-conjugated anti-mouse secondary antibodies (Millipore, Burlington, MA) were used to detect TH immunoreactivity in association with chemiluminescence-based detection, ECL-Plus (Thermo Fisher Scientific, Waltham, MA).