Aliquots of the cell extracts were taken for protein determination. Pre cleaned mobile extracts containing 200 g of protein were incubated with rabbit polyclonal anti IGF I receptor subunit antibody over night at 4 C with continuous rocking. After the addition of 30 l of 50% Protein G Plus/Protein A agarose slurry, the products were further incubated at 4 C for 3 h. The beans were then washed four times with RIPA buffer and mixed with electrophoresis sample buffer. Male Sprague?Dawley mice andmale CD1 rats weremaintained in a 1-2 h light/dark cyclewith MAP kinase inhibitor food andwater. Experiments were conducted in accordance with the European Communities Council Directive of November 2-4 1986 and with the axioms of Laboratory Animal Care in Italy. Nucleus accumbens was isolated from 300 m thick coronal mind slices by microdissection as previously described. Nucleus accumbens slices were incubated in a recently oxygenated Krebs Hepes buffer containing 25 mM Hepes/NaOH, 10 mM glucose, 125 mM NaCl, 3. 8 mM KCl, 1. 2 mM MgSO4, 1. 2 mM CaCl2, 1. 2 mMKH2PO4 at 2-8 C for 60?90 minimum. Afterwards, the tissue slices were incubated in the same buffer Metastatic carcinoma at 28 C and confronted with both naltrindole or vehicle for 5 min, accompanied by 20 min exposure to NDMC or vehicle. The medium was aspirated and 200 l of ice cold RIPA buffer was added. The samples were homogenized by sonication and located at?80 C. CD 1 mice were treated with naltrindole or saline 15 min prior to the administration of either car or NDMC. NDMC was dissolved in glacial acetic acid and the answer was buffered to pH 5. 5. The animals were sacrificed by cervical dislocation 30 min after NDMC management. The brain was rapidly eliminated and as indicated above nucleus accumbens was dissected from brain coronal pieces. Structure from each animal was collected in 300 l of ice-cold RIPA buffer, sonicated and kept at?80 C. Aliquots of the tissue extracts were taken for protein determination. Aliquots of cell or tissue extracts containing equal quantity of protein were subjected to SDS polyacrylamide gel electrophoresis and the proteins were electrophoretically Flupirtine utilized in polyvinylidene difluoride membranes. The efficiency of the transfer was controlled by gel staining and by following the transfer of pre stained protein standards. Non-specific binding internet sites were blocked by incubation in 20 mM Tris?HCl, 137 mM NaCl and 0. 05% Tween 20 containing 53-56 BSA for 1 h. After washing with TBS T barrier, the membranes were incubated over night at 4 C with one-of the following antibodies: anti phospho Ser9 GSK 3, anti phospho Thr308 Akt, anti GSK 3, anti Akt anti phospho IGF I receptor /insulin receptor, anti IGF I receptor and anti phosphotyrosine.