| OCR Text |
Show 16 nucleus accumbens tissue than striatal tissue was subjected to SOS-PAGE in order to have approximately equal levels of OAT monomer immunoreactivity in the saline control lanes. For data presented in Figure 1.2, equal volumes of sample were loaded into each well. Samples were then transferred overnight to a polyvinylidene difluoride (PVOF) hybridization Elmer Life Sciences, Waltham, with MA). transfer membrane (Perkin Each membrane was blocked for 30 min Starting Block Blocking Buffer (Pierce Biotechnology, Rockford, IL). The membrane was then incubated for 1 h at room temperature or overnight at 4 'C with a N-terminal OAT antibody (a generous gift from Dr. Roxanne Vaughan, University of North Dakota (Freed et aI., 1995)). The PVDF membrane was then washed 5 times in Tris-buffered saline with Tween (T8ST; 250 mM NaCI, 50 mM Tris, pH 7.4, and 0.05% Tween 20). The membranes were then incubated for 1 h with a goat anti-rabbit horseradish peroxidase conjugated secondary antibody (Biosource International, Camarillo, CA). bands were visualized Plus (Perkin-Elmer densitometry using Western Lightning Chemiluminescence Reagents Life using a Following five washes in TBST, the Sciences, Waltham, FluorChem Corporation, San Leandro, CA). SP DAT MA) Imaging and were System immunoreactivity quantified by (Alpha with an Innotech apparent molecular weight greater than approximately 120 kilodalton (kDA) was defined as OAT complexes. Protein concentrations were determined using the method of Lowry et al. (1951). Synaptosomal [3H]DA uptake. [3H]DA uptake was evaluated by incubating retained striatal synaptosomes (as described above) at 37 'C for 10 |
