Because cells in RIPA buffer supplemented with . Triton X . Following incubation for min at ice bath temperature, cells were sonicated for s in ice bath and centrifuged at , g for min at C. Aliquots in the cell extracts were taken for protein determination. Pre cleared cell extracts containing g of protein have been incubated with rabbit polyclonal anti IGF I receptor subunit antibody overnight at C with constant rocking. Following the addition of l of Protein G Plus Protein A agarose slurry , the samples have been even further incubated at C for h. The beads were then washed four occasions with RIPA buffer and mixed with electrophoresis sample buffer Animals and tissue dissections Male Sprague Dawley rats andmale CD mice weremaintained within a h light dark cyclewith food andwater ad libitum. Experiments had been carried out in accordance together with the European Communities Council Directive of November and together with the concepts of Laboratory Animal Care in Italy .
Nucleus accumbens was isolated from m thick coronal brain slices by microdissection as previously described Tissue slice treatment method Nucleus accumbens slices were incubated within a freshly oxygenated Krebs Hepes buffer containing NU7441 mM Hepes NaOH, mM glucose, mM NaCl, mM KCl mM MgSO mM CaCl mMKHPO at C for min. Thereafter, the tissue slices had been incubated in the similar buffer at C and exposed to both naltrindole or car for min, followed by min publicity to NDMC or motor vehicle. The medium was aspirated and l of ice cold RIPA buffer was added. The samples were homogenized by sonication and stored at ? C In vivo treatment method CD mice were handled with naltrindole or saline min just before the administration of either vehicle or NDMC . NDMC was dissolved in glacial acetic acid as well as the choice was buffered to pH The animals had been sacrificed by cervical dislocation min just after NDMC administration. The brain was quickly eliminated and nucleus accumbens was dissected from brain coronal slices as indicated above. Tissue from every single animal was collected in l of ice cold RIPA buffer, sonicated and stored at ? C.
Aliquots within the tissue extracts have been taken for protein determination Western blot analysis Aliquots of cell or tissue extracts containing equal volume of protein had been subjected to SDS polyacrylamide gel electrophoresis and the proteins were electrophoretically Rutaecarpine transferred to polyvinylidene difluoride membranes . The efficiency in the transfer was controlled by gel staining and by following the transfer of pre stained protein requirements. Nonspecific binding online websites were blocked by incubation in mM Tris HCl, mM NaCl and . Tween containing BSA for h. Following washing with TBS T buffer, the membranes were incubated overnight at C with 1 within the following antibodies: anti phospho Ser GSK , anti phospho Thr Akt , anti GSK , anti Akt anti phospho IGF I receptor insulin receptor , anti IGF I receptor and anti phosphotyrosine .