| OCR Text |
Show 60 synaptic proteins, and thereby provide novel insights into the function and regulation of a variety of membrane-associated proteins including OAT. Methods Animals. Laboratories, Male Sprague Dawley Raleigh, NC) rats maintained were (300-450 g; under Charles controlled lighting and Rats were temperature conditions, with food and water provided ad libitum. sacrificed River by decapitation and striata were dissected and quickly placed in ice cold 10 mM HEPES, 0.32 M sucrose, pH 7.4, and 10 mM NEM until processing. All procedures were conducted in accordance with National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and approved by the University of Utah Institutional Animal Care and Use Committee. Immunoprecipitation. Striata were homogenized in ice-cold 10 mM * g; 5 min; HEPES, 0.32 M sucrose, pH 7.4, 10 mM NEM, and centrifuged (1,000 . 40 C). Since the DAT contains alkylating agent, NEM, was added to the spurious disulfide bond formation. * multiple cysteine residu es, the free-sulfhydryl homogenization buffer to prevent The supernatant was then centrifuged (16,000 g; 20 min; 40 C) to yield the crude synaptosome pellet. The resultant pellet was resuspended at 100 mg/ml original wet weight in 10mM HEPES, 300mM NaCI, pH 7.4, 1 % Triton-X 100, 1 mM PMSF, and 1 :200 protease inhibitor cocktail (Sigma-Aldrich). Following a 1-h incubation at 4'C with repeated inversion, the was pooled into 1-ml aliquots. * g; 20 min; 40 C). The supernatants were Protein A- and G-linked magnetic beads (Dynal centrifuged (16,000 mixture Biotech) were pre-washed with 10mM HEPES, 300mM NaCI, 1 % Triton-X 100, |
