Ir 790

Total proteins were extracted by dissolving cells and synaptosomes in the solubilizing buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 0.5% Triton X-100, 2 mM phenylmethylsulphonyl fluoride PMSF,1mMDTT, 0.1% SDS) with protease inhibitor (Amersham Biosciences, Milan, Italy) and phosphatase inhibitor (cocktail II and III; Sigma Aldrich, Milan, Italy). Protein samples (30 µg) were submitted to 10% SDS PAGE and transferred onto nitrocellulose filters. The Western blot was incubated with anti-TOM40 (1:1000; Cell Signaling, Boston, USA), anti-Cytochrome C (1:1000; Cell Signaling, Boston, USA), anti-OPA1 (1:1000; Cell Signaling, Boston, USA), anti-FIS1(1:1000; Cell Signaling, Boston, USA), anti-synaptophysin (1:2000; Cell Signaling, Boston, USA), PSD95(1:1000; Cell Signaling, Boston, USA), anti-βActin (1:1000; SIGMA) antibodies. Primary antibody was detected by the Odyssey scanner (L-Licor) and using secondary antibody labeled with IR 790, (1:10,000; Life Technology) according to the manufacturer's instructions. Band intensities were analyzed with ImageJ and expression was adjusted to βActin expression. The protein levels were expressed as intensity relative to control.