Animals and treatments
Animal maintenance and experimentation were conducted in strict
compliance with the European and National Law for Laboratory Animal use
(Directive 2010/63/EU and Greek Law 161/91), the FELASA recommendations
and the ethical and practical guidelines for the care and use of
laboratory animals set by the competent veterinary services of Athens.
All experiments were carried out in wild-type C57BL/6J mice.
Three-month-old male mice were divided into two groups (n=4/ group) and
injected intraperitoneally once per day with saline or 5 mg/kg U50,488H
for 6 consecutive days. The mice were sacrificed 3 h after the last
U50,488H/saline injection and the hippocampus, cortex, and striatum were
rapidly dissected on ice. Isolated regions were placed in cold PBS and
immediately homogenized by sonication in RIPA buffer containing 50 mM
Tris-HCl pH 7.2, 150 mM NaCl, 2 mM EDTA, 1% Triton 100-X, 1 % sodium
deoxycholate, 0.5 % SDS and 1 mM dithiothreitol in the presence of
protease inhibitors and incubated for 1 h at 4 οC. The
resulting supernatant was collected after centrifugation at 8,000 x g
for 20 min.