Anti cypd

The brain tissues of mice and cells were homogenized in lysis buffer containing protease inhibitors. The homogenate was centrifuged at 14000 g for 15 min at 4°C and the protein concentration was determined using the BCA kit. 30 μg lysate was loaded onto 10% SDS-PAGE. The proteins were transferred to PVDF membranes (Millipore, MA, USA). The membranes were blocked for 1 h in 5% dry milk and then incubated overnight with one of the following primary antibodies: anti-iNOS (ab178945), anti-Pro-caspase-1 (ab179515), anti-ANT (ab102032), anti-Cyp D (ab16045) (Abcam, Cambridge, MA, USA), anti-TH (#2792), anti-COX2 (#12282), anti-p-p65 (#3033), anti-Cleaved-caspase-1 (#89332), anti-Cyto C (#4280), anti-VDAC (#4866), anti-COX4 (#4850), or anti-β-actin (#3700) (Cell Signaling Technology, Beverly, USA). After washing 3 times in TBST for 5 min each, the membranes were incubated with goat anti-mouse, anti-rabbit, or anti-rat HRP for 1 h at room temperature. Then, the membranes were washed 3 times in TBST for 5 min each. The signal was visualized using an ECL chemiluminescence kit (Amersham Biosciences/GE Healthcare; Piscataway, NJ).

Frozen-thawed mitochondrial pellets were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Separated proteins were transferred to a methanol-activated polyvinylidene difluoride membrane. Immunoblotting was performed as recommended by the manufacturers of the antibodies. Mouse monoclonal anti-cyclophilin D (cypD; Mitosciences, Eugene, OR, USA), rabbit polyclonals anti-OGDH, anti-DLST, anti-DLD, anti-VDAC1, anti-CypD, anti-GR, anti-GPX1, anti-TRX, and anti-PRX3 (Abcam, Cambridge, UK) primary antibodies were used at concentrations of 1 microg/mL, while rabbit polyclonal anti-manganese superoxide dismutase (MnSOD; Abcam) at 0.2 microg/mL. Immunoreactivity was detected using the appropriate peroxidase-linked secondary antibody (in 1:4000 dilution, donkey anti-mouse or donkey anti-rabbit; Jackson Immunochemicals Europe Ltd., Cambridgeshire, UK) and enhanced chemiluminescence detection reagent (ECL system; Amersham Biosciences GE Healthcare Europe GmbH, Vienna, Austria).

The cultured cells were seeded onto six-well plates, and their differentiation potency was characterized and tested using immunofluorescence staining analysis. To fix the cells, 4% paraformaldehyde (PFA) was used. Mouse monoclonal anti-nestin antibody (1:500, Abcam, Boston, MA, USA), rabbit polyclonal anti-glial fibrillary acidic protein (GFAP) antibody (1:500, Abcam), and mouse monoclonal anti-β-tubulin III antibody (1:500, Abcam) were used for NPCs identification. D4′, 6′-diamidion-2′-phenylindole (DAPI, 1:1000, Abcam) was used to counterstain the nuclei. To detect CypD and Complex V (mitochondrial marker), anti-CypD (1:200, ab110324, Abcam) and anti-complex V (1:200, 45, 9000, Invitrogen, Carlsbad, CA, USA) antibodies were used. After fixing the cells for 20 min, they were placed in 0.1% Triton X-100 for 30 min, followed by a blocking buffer (10% goat serum) for 1 h. The cells were then incubated with primary antibodies overnight at 4 °C. Samples were incubated in secondary antibodies [fluorescein isothiocyanate (FITC) or tetramethylrhodamine isothiocyanate (TRITC)-conjugated IgG] for 2 h, followed by DAPI incubation for 5 min at room temperature. The slides were rinsed with DPBS, and the images were captured using a laser confocal microscope (TSC SP2, Leica, Manheim, Germany).