Anti fis1

Total cell extracts or nuclear extracts were separated by SDS-PAGE and transferred to PVDF membranes. The following antibodies were used for immunoblot analysis: anti-HK2, anti-PFKM, anti-LDHa1, anti-PDK2, anti-PDHa1, anti-ATP5B, anti-IDH2, anti-HIF1α, anti-MnSOD, anti-Cu/ZnSOD, anti-Catalase, anti-GR, anti-GRX1, anti-GPX1, anti-PRX3 and anti-TRX2 antibodies and secondary antibody were purchased from Proteintech. anti-MnSOD (acetyl K68) antibody was purchased from Abcam. Anti-β-actin, anti-ComplexV, anti-Lc3B, anti-MFN1, and anti-Fis1 antibodies were from Cell Signaling Technology. Protein expression is visualized on Tanon-5200 Chemi-luminescent Imaging System (Tanon Science Technology).

Total protein, cytoplasmic protein and mitochondrial protein were separated by SDS-PAGE and transferred to nitrocellulose membranes (Millipore) followed by blocking and immunoblotting with various primary antibodies, including anti-RCAN1, anti-FLAG (Sigma), anti-COX4, anti-Drp1, anti-Fis1, anti-Mfn2, anti-Opa1 (all Cell Signaling Technology, Boston, U.S.A.), anti-fibronectin (Millipore), anti-collagen I, anti-β-tubulin, anti-β-actin (Santa Cruz Biotechnology, Dallas, U.S.A.). After overnight incubation at 4°C, the membranes were immersed in a solution including appropriate secondary antibodies (Santa Cruz) for 1 h at room temperature. The blots were developed using ECL kit (Millipore).

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.