In the mammalian genome, DNA methylation is the most common and well-characterized epigenetic mark, which consists of the covalent addition

en analyzed by western blot with goat anti-calnexin, mouse anti-b1-integrin, rabbit anti-porin and goat anti-ANT primary antibodies, respectively. To verify the amount of loaded proteins some filters were blotted with mouse anti b-tubulin antibody. Membrane and mitochondrial proteins from rat tissues and cells were immunoprecipitated by using commercially available mouse monoclonal IgG antibodies directed against EAAC1 or NCX1 . To recover the immunocomplexes, the samples were incubated with A-Sepharose beads. This method has been already used to analyze protein complex from mitochondria. The following primary antibodies were used to blot the membranes: mouse anti-EAAC1 , rabbit anti-GLAST and rabbit anti-GLT1, mouse anti-NCX1, anti-NCX2 Materials and Methods Isolation ” of plasma membranes and mitochondria from rat tissues and cell cultures Mitochondrial NCX1/EAAC1 Sustain Brain Metabolism monoclonal IgM antibody, rabbit anti-NCX3, and goat anti-ANT, goat anti-AGC2. A ChemiDoc station and the Quantity one analysis software were used for signal detection and analysis. Tissue mitochondria immunohistochemistry Mitochondria derived from rat hippocampus or cortex by discontinuous Percoll gradient were spotted onto poly-Llysine-coated micro slides by cytocentrifugation. Half of the preparation was then loaded with 2 mM Red MitoTracker for 15 min. Then, mitochondria were fixed in 4% PFA, permeabilized in PBS with 10% normal rabbit serum and 0.1% Triton for 1 h at room temperature, washed in PBS and incubated MedChemExpress Acacetin overnight at 4uC with the primary antibodies goat anti-EAAC1 and/or mouse anti-NCX1 in PBS with 1% normal rabbit serum. After washing, mitochondria were incubated with antimouse Alexa Fluor 488 dye-conjugated antibody or antigoat Alexa Fluor 555 dye-conjugated antibody at room temperature for 1 h. The fluorescent signal was visualized using an LSM 510 laser scanning confocal microscope equipped with an Argon and a HeNe laser. EAAC1 pre-embedding immunoelectron microscopy Adult male Wistar rats were anesthetized with 12% chloral hydrate and perfused transcardially with 4% paraformaldehyde and 0.5% glutaraldehyde in 0.1 M phosphate buffer, or nonimmune serum as negative control and then sections were incubated in the appropriate goat biotinylated secondary antibodies; diaminobenzidine was used as chromogen. After completion of immunohistochemical staining, sections were washed in PB, incubated in 2.5% glutaraldehyde, washed in PB and postfixed in 1% OsO4. After dehydration in ethanol and infiltration in Epon-Spurr resin, sections were flat-embedded between acetate sheets and polymerized at 60uC for 48 h. The embedded sections were then examined under a dissecting microscope. Small tissue strips containing the cerebral cortex or the hippocampus were cut out, glued to blank epoxy and sectioned with an MTX ultramicrotome. Thin sections were stained with lead citrate and examined in a Philips CM10 transmission electron microscope. baseline values before stimulation. In a separate set of ” experiments, cells were loaded with 10 nM TMRE at 37uC in culture medium. Prior to the measurements cells were then permeabilized for 10 min with digitonin 5 mM in “intracellular”buffer with the following composition: 5 NaCl, 100 KCl, 70 mannitolo, 25 sucrose, 10 KH2PO4, 0.0001 CaCl2, 10 Tris-HCl, 1 MgCl2, 5,5 glucose, 2 pyruvate, 2 malate, 3 succinate, 0.1 ADP, 0.005 digitonin, 0.00001 TMRE, pH 7.3/KOH. Digitonin has been already used to selectively permeabilize