Animals and social conditions
All procedures were conducted by the guidelines of Ilia State University Research Project Ethics Commission. Male C57BL/6 J wild-type and IL-10-deficient C57BL/6 J mice (mice homozygous for the IL10tm1Cgn targeted mutation, 8–12 weeks of age) were purchased from Jackson Laboratories (Bar Harbor, ME, USA) and provided by the Institute of Medical Biotechnology of Tbilisi State Medical University (Tbilisi, Georgia). These IL-10 mutant mice are viable and fertile under specific pathogen-free conditions. IL-10-deficient phenotype is associated with altered lymphocyte and myeloid profiles, elevated serum amyloid. A levels, altered responses to inflammatory or autoimmune stimuli, increased depressive-like behavior [30] and decreased neuroprotection [31].
Preparation of membrane fractions
The brain cortex was removed and gently homogenized in isolation buffer (50 mM HEPES, pH 7.5, 125 mM NaCl, 2 mM KCl, 100 mM sucrose, 1,8 mM CaCl2, 1 mM MgCl2, 5 U/mL aprotinin). Homogenate was centrifuged at 1250×g for 5 min. Obtained supernatant was immediately centrifuged at 21,000×g for 20 min and membrane fractions were used for further experiments.
Coimmunoprecipitation and immunobloting
Membrane fractions were resuspended in ice-cold solubilization buffer (20 mM Tris–HCl pH 8.0, 137 mM NaCl, 10 % glycerol, 1 % Triton X-100, 2 mM EDTA), incubated for 30 min at 4 °C. The unsolubilized material was removed by centrifugation (60 min at 20,000×g). Anti-SigR1 antibody (rabbit polyclonal OPRS1, Abcam 53852, UK) and prewashed Protein A/G-agarose resin (Pierce) were added to the supernatant at 5 μg of antibody per 400 μg of protein and incubated overnight at 4 °C. After washing (12,000×g, 20 min), the protein A/G-Agarose pellets were resuspended in 100 mM glycine, pH 3.0, for 10 s, and then a pretitrated volume of 1.0 M Tris, pH 9.5, was added to adjust the pH to 7.4. Protein complexes in the supernatants (1000×g, 12 min) were then analyzed by Western blotting.
For immunoblotting experiments, 50 μg of protein was separated by SDS–polyacrylamide gel electrophoresis and transferred to nitrocellulose sheets. After blocking with blocking buffer (5 % bovine serum albumin, 0.05 % Tween 20 in Tris–HCl-buffered saline), the sheets were incubated either with BiP rabbit polyclonal BiP antibody (Abcam, UK), with Rac1 rabbit polyclonal antibody (Santa Cruz, USA), with rabbit monoclonal inositol 1,4,5-trisphosphate receptor (IP3R) antibody, (Abcam, UK), or with goat polyclonal NR2B glutamate receptor (ε2) antibody (Santa Cruz, USA). As a loading control, β-actin expression levels were measured by goat polyclonal anti-actin antibody (Santa Cruz, USA). The nitrocellulose membranes were washed three times with wash buffer (150 mM NaCl, 10 mM Tris–HCl, pH 7.0, and 0.05 % Tween 20) containing 5 % skim milk. And then incubated with horseradish peroxidase-conjugated goat-anti-rabbit or rabbit-anti mouse-IgG antibody. Labeled bands were visualized using enhanced chemiluminescence (Amersham, California, USA) and analyzed by densitometric scanning. The content of proteins was quantified by the intensity of the bands, which is linear to the quantity of samples applied to the gel. The protein levels were normalized against β-actin intensity.
Protein concentration was determined using a dye-binding method (Bio-Rad).
NADPH oxidase activity
NADPH oxidase activity was measured in mice brain according to O’Brien et al. 2009 [32]. The membrane fractions were resuspended in 50 mM phosphate buffer at pH 7.4. Paired assays were conducted by incubation of 20–60 μg of membrane proteins with 10 mM phosphate buffer, 130 mM NaCl, 1 mM EGTA, 10 μM flavin adenine dinucleotide (FAD), 2 mM NaN3, 50 μM oxidized cytochrome c, and 10 μM GppNHp (Guanosine 5′-[β,γ-imido]triphosphate). In some experiments 100 μM glutamate, 1 μM (+)-pentazocine, 1 μM haloperidol, 1 μM 4-PPBP or 1 μM BD1063 were added. One reaction of each pair contained 200 units of superoxide dismutase (SOD; Sigma–Aldrich). Reactions were initiated by the addition of NADPH to a final concentration of 200 μM. After 1 h, activity was measured as the SOD-inhibitable increase in absorbance at 550 nm. Results of the NOX activity are expressed as the reduced cytochrome c per milligram protein per min as relative absorbance units at 550 nm (RAU at 550 nm).
Statistical analysis
All experiments were performed in triplicates and repeated at least twice. Results are expressed as mean ± the standard error of the mean (SEM). For statistical analysis, a t test was used. A difference between experimental groups was considered to be significant when P < 0.05.