The electrophoretic mobility shift assay was performed as described previously . The consensus sequence-specific oligo-nucleotide probes were end-labeled with γ-32P-ATP
according to the manufacturer’s recommendations. The oligonucleotide with the C/EBP consensus binding sequence used were 5′-GGTTCTTGCGCAACTCACTGAA-3′ and 3′-TTCAGTGAGTTGCGCAAGAACC-5 For SRT1720 purchase the binding reaction, 2 ng labeled oligonucleotide (approximately 20 000 cpm) and 2 μg poly dIdC (Amersham Pharmacia Biotech) carrier were incubated with 2 μg nuclear protein in a binding buffer (10 mM HEPES, 60 mM KCl, 1 mM DTT, 1 mM EDTA, 7% glycerol, and pH 7.6) for 30 min at room temperature. DNA–protein complexes were resolved on 6% nondenaturing polyacrylamide gels and visualized by exposure to autoradiographic films. Sprague-Dawley rats (230–250
g) were anesthetized by i.p. injection of chloral hydrate (400 mg/kg), positioned in a stereotaxic apparatus, and either LPS (from Salmonella enteritidis; Sigma, St. Louis, MO), IL-13, IL-13 antibody, or a combination of 2–3 were stereotactically injected into the right cerebral cortex (AP+4.8 mm ML, −5.5 mm, DV −6.0 mm from the bregma) according to Paxinos’ atlas. selleck chemicals llc The animals were categorized into to five groups: group I, PBS injection (30 μL); group II, LPS injection (10 μg); group III, IL-13 (100 μg) injection; group IV, LPS (10 μg) + IL-13 (100 μg) injection; and group V, LPS (10 μg) + IL-13 (100 μg) + IL-13 neutralized antibody (NA, 10 ng) in a final volume of 30 μL PBS injected at a rate of 0.15
μL/min using a 26-gauge Hamilton syringe attached to an automated pump Grape seed extract and left in situ for an additional 5 min to avoid reflux along the injection tract. A 1.5 m diameter, 45 cm deep Morris water maze was filled with water to a depth of 26.5 cm. The water temperature was kept at 26 ± 2˚C. A circular platform, 25 cm high, and 12 cm in diameter was placed into the tank at a fixed location in the centre of one of four imaginary quadrants. Approximately 1.5 L of milk was used to make the water opaque. The rat was then guided to swim to the platform. Activity in the water maze was recorded using a CCD camera on the ceiling above the centre of pool, which was attached to an automated tracking system (Noldus, Netherlands). A single experiment was performed with three rats. Behavioral measures included latency to targets, swing speed (cm/s), number of platform crosses, and percent time within the targeted area. Percent time in appositive object in reversal trial and in targeted object in extinction test was also conducted. Data were analyzed by Etho Vision 3.1. The animals were transcardially perfused with a saline solution containing 0.5% sodium nitrate and heparin (10 U/mL), followed by 4% paraformaldehyde dissolved in 0.1 M phosphate buffer (PB).