Frozen samples (0.25-0.50 g of tissue) were homogenized in 20 volumes of boiling 1% wt/vol sodium dodecyl sulfate containing 1 µg/mL of leupeptin(Chemicon International, Inc, Temecula, Calif), sonicated, and boiled for5 minutes. The samples were centrifuged at 1000g for 5 minutes; the protein concentration in the supernatants was determined by the bicinchoninic acid method (Pierce Biotechnology, Inc, Rockford, Ill). Protein (100 µg per lane) was subjected to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, as described.5 Samples from each pair of subjects and matched control samples were run in quadruplicate on the same gel to minimize variations in transfer and immunolabeling. The proteins were transferred to 0.2-µm nitrocellulose (Schleicher & Schuell Bioscience, Inc, Keene, NH) at 200 mA for 18 hours.25 After transfer of the proteins onto nitrocellulose, the gels were stained with Coomassie blue to visualize protein loading and transfer efficiency. If this assessment demonstrated protein degradation in any sample, the sample and its match were not included in the analysis. All blotting steps were performed at room temperature in blotting buffer (a combination of 86mM sodium phosphate, pH 7.3; 150mM sodium chloride; 0.05% vol/vol polyethylenesorbitan monolaurate[Tween 20] [Sigma-Aldrich Corp, St Louis, Mo]; and 0.02% wt/vol sodium azide). Nonspecific binding was blocked by incubation in blotting buffer containing2.5% wt/vol nonfat dry milk (Carnation) for 8 hours. After 2 rinses (15 minutes each), blots were cut into sections containing the proteins of interest. The blot sections were incubated for 2 hours with primary antibodies to DARPP-32(mouse monoclonal antibody C24-6a, 1:1000 dilution), synapsin I (rabbit polyclonal antibody G-472 [1:200 dilution], G454/455 [1:2000 dilution], or G486 [1:2000 dilution]), or the α subunit of calcium/calmodulin-dependent protein kinase II (CaMK IIα) (rabbit polyclonal antibody RU16, 1:1000 dilution). None of the antibodies used was selective for the phosphorylation state of the proteins, ie, phosphorylated and dephosphorylated forms should be detected equally. After 2 rinses (15 minutes each), the DARPP-32 blot sections were incubated for 1 hour with rabbit anti–mouse IgG (1:500 dilution) (Pierce Biotechnology, Inc). After 2 rinses (15 minutes each), all blotsections were incubated for 90 minutes in iodine 125 labeled protein A (1:1000 dilution)(Amersham Holdings, Inc, Arlington Heights, Ill). After two 15-minute and two 5-minute rinses, the blot sections were dried, wrapped in plastic, and subjected to imaging analysis (PhosphorImager; Molecular Dynamics, Sunnyvale, Calif).