Phosphatase inhibitor mixture 2 and 3

Striatum and ventral midbrain from injected and noninjected hemispheres were rapidly dissected and homogenized with six volumes of Triton lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 5% glycerol, 1% Triton X-100, 1 mM EDTA, 1× EDTA-free Complete protease inhibitor mixture [Roche] and 1× phosphatase inhibitor mixture 2 and 3 [Sigma]). Tissues were disrupted using a mechanical homogenizer (IKA T10 basic, Ultra Turrax). The Triton-soluble fraction was obtained after ultracentrifugation at 100,000 × g for 30 min at 4 °C. The resulting pellets were further extracted by sonication in 3× volumes of SDS lysis buffer (50 mM Tris⋅HCl pH 7.4, 2% SDS, 1× EDTA-free Complete protease inhibitor mixture [Roche] and 1× phosphatase inhibitor mixture 2 and 3 [Sigma]). Samples were centrifuged at 21,000 × g for 30 min at 25 °C to obtain the Triton-insoluble (SDS-soluble) fraction. Protein concentration was determined using the BCA assay (Pierce Biotech).