Phosphate inhibitor cocktails 1 and 2

Mice were euthanized by cervical dislocation. Mouse brain subregions (ventral midbrain, VM) were located following procedures described previously [23 (link)]. Mouse brain tissues were homogenized in lysis buffer [10 mM Tris–HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40, 10 mM Na-β-glycerophosphate, Phosphate Inhibitor Cocktails I and II (Sigma), and a complete protease inhibitor mixture (Roche)] using a Diax 900 homogenizer. Five milliliters of lysis buffer per gram of brain tissue was used for homogenization. After homogenization, samples were rotated at 4 °C for 30 min to ensure complete lysis. The homogenates were then centrifuged at 52,000× g (rcf) for 20 min, and the resulting supernatants were collected. Protein levels were quantified using the BCA Protein Assay Kit (Pierce) with BSA standards. Proteins were then subjected to immunoblotting with the antibodies of interest. Immunoreactive bands were visualized with an enhanced chemiluminescence kit (Pierce). Densitometric analyses of protein bands were performed using ImageJ (NIH, http://rsb.info.nih.gov/ij/).

Mice were euthanized by cervical dislocation. Mouse brain subregions (CTX, VM, STR) were located following procedures described previously [45 (link)]. The frontal cortex was dissected and designated as CTX. Mouse brain tissues were homogenized in lysis buffer [10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40, 10 mM Na-b-glycerophosphate, Phosphate Inhibitor Cocktails I and II (Sigma), and a complete protease inhibitor mixture (Roche)] using a Diax 900 homogenizer. After homogenization, samples were rotated at 4°C for 30 min to ensure complete lysis. The homogenates were then centrifuged at 52,000 rpm for 20 min, and the resulting supernatants were collected. Protein levels were quantified using the BCA Protein Assay Kit (Pierce) with BSA standards. Proteins were then subjected to immunoblotting with the antibodies of interest. Immuno-reactive bands were visualized with an enhanced chemiluminescence kit (Pierce). Densitometric analyses of protein bands were performed using ImageJ (NIH, http://rsb.info.nih.gov/ij/).