Rats were administered 10 pmol or 1 nmol enzastaurin, or vehicle 0.5, 3, or 18 hr prior to an injection of 3 mg/kg AMPH
(s.c.). The rats were euthanized 10-30 min following AMPH and the ventral striatum was dissected. The tissue
was immediately frozen in liquid nitrogen, then 250 μl boiling 1% SDS was added to each sample. The samples were sonicated
for 5 pulses at frequency of 20 kHz, amplitude 50% (sonic dismembrator, Fisher Scientific, Pittsburgh, PA) and then spun at 14,000
rpm at 4°C, saving the supernatant. The samples (75 μg) were separated by SDS-PAGE on a 12% polyacrylamide gel. The
proteins were transferred to a nitrocellulose membrane at 100 mA for 16 hr. The membranes were blocked in a buffer (5% w/v milk,
150 mM NaCl, 10 mM Tris, 0.05% Tween20) before probing for rabbit anti-phosphoser41-growth associated protein 43
(pGAP43) (Santa Cruz Biotechnology Inc., Santa Cruza, CA) and goat anti-GAP43 (Santa Cruz Biotechnology) antibodies for 24 hr at
4°C on two separate membranes. Primary antibody binding was detected with secondary antibodies for 1 hr at room
temperature: antibodies for goat anti-rabbit for pGAP43 and donkey anti-goat for total GAP43 (Santa Cruz Biotechnology). The
antibodies were imaged with Chemiluminescent Western Substrate (EMD Millipore, Darmstadt, Germany) and band densities were
quantified using Image J software.
(s.c.). The rats were euthanized 10-30 min following AMPH and the ventral striatum was dissected. The tissue
was immediately frozen in liquid nitrogen, then 250 μl boiling 1% SDS was added to each sample. The samples were sonicated
for 5 pulses at frequency of 20 kHz, amplitude 50% (sonic dismembrator, Fisher Scientific, Pittsburgh, PA) and then spun at 14,000
rpm at 4°C, saving the supernatant. The samples (75 μg) were separated by SDS-PAGE on a 12% polyacrylamide gel. The
proteins were transferred to a nitrocellulose membrane at 100 mA for 16 hr. The membranes were blocked in a buffer (5% w/v milk,
150 mM NaCl, 10 mM Tris, 0.05% Tween20) before probing for rabbit anti-phosphoser41-growth associated protein 43
(pGAP43) (Santa Cruz Biotechnology Inc., Santa Cruza, CA) and goat anti-GAP43 (Santa Cruz Biotechnology) antibodies for 24 hr at
4°C on two separate membranes. Primary antibody binding was detected with secondary antibodies for 1 hr at room
temperature: antibodies for goat anti-rabbit for pGAP43 and donkey anti-goat for total GAP43 (Santa Cruz Biotechnology). The
antibodies were imaged with Chemiluminescent Western Substrate (EMD Millipore, Darmstadt, Germany) and band densities were
quantified using Image J software.