P ser40 th

Striatal slices of mouse brain (300 μm, Leica Vibratome) were preincubated in Krebs buffer (118 mM NaCl, 4.7 mM KCl, 1.5 mM Mg₂SO₄, 1.2 mM KH₂PO₄, 25 mM NaHCO₃, 11.7 mM glucose, 1.3 mM CaCl₂) at 30 °C under constant oxygenation (95% O₂/5% CO₂) for 60 min, with a change of buffer after 30 min. They were then treated with MDMA for 5 min. The buffer was then removed, and the slices were rapidly frozen, sonicated in 1% SDS and boiled for 10 min. Amounts of protein in homogenates were determined using the bicinchoninic acid protein assay method (Pierce, Rockford, IL, USA). Equal amounts of protein were processed by using 10% acrylamide gels. Western blot (WB) was carried out with phosphorylation state-specific antibodies against P-Ser¹⁹-TH, P-Ser³¹-TH and P-Ser⁴⁰-TH (Millipore, Solna, Sweden) or antibodies that are not phosphorylation state-specific against total TH and actin (Millipore). Antibody binding was detected by enhanced chemiluminescence (ECL; GE Healthcare, Uppsala, Sweden) and quantified by densitometry (NIH IMAGE 1.61 software). Data are percentage of total levels.

Immunoblotting was performed as described earlier (Qi et al., 2009 (link)). Frozen tissue samples were sonicated in 1% SDS, transferred to Eppendorf tubes and boiled for additional 10 min. Small aliquots of the homogenate were retained for protein determination using the bicinchoninic acid protein assay method (Pierce, Rockford, IL, United States). Equal amounts of protein (20 μg) were loaded onto 12% acrylamide gels, and the proteins were separated by SDS-PAGE and transferred to Immobilon^®-P Polyvinylidene Difluoride membranes (Sigma). Immunoblotting was performed on the membranes using P-Ser¹⁹-TH (Merck Millipore, Billerica, MA, United States), P-Ser³¹-TH (Millipore), P-Ser⁴⁰-TH (Millipore), P-Ser⁸⁴⁵-GluA1 (UBI), and antibodies, which are not phosphorylation state-specific to estimate total levels of TH (Millipore) and GluA1 (UBI). The antibody binding was detected by incubation with goat anti-mouse or anti-rabbit horseradish peroxidase-linked IgG (1:6000–8000 dilution) and detected using ECL immunoblotting detection reagents (GE Healthcare, Little Chalfont, United Kingdom).