Cultured SK-N-MC neuroblastoma cells were grown in MEM medium supplemented with 5 nM rotenone, a pesticide.
Synuclein and ubiquitin levels were determined by immunocytochemistry and protein dot blots. For immunocytochemistry, control and rotenone-treated cells were fixed and incubated with primary antibodies, polyclonal rabbit anti-alpha synuclein 1:100 (gift of Mark Cookson, Bethesda MD) and polyclonal rabbit against ubiquitin, for 24 hr, followed by 2nd biotinylated ab and ABC Elite kit.
For dot blots, control and rotenone-treated cells were lysed in lysis buffer (Promega, Madison, WI) containing 0.5 mg/ml benzamidine, 2 µg/ml aprotinin, 2 µg/ml leupeptin, 0.75 mM phenylmethylsulfonyl fluoride, 700 U/ml DNase I, and 1% mercaptoethanol. Primary antibodies were anti-alpha synuclein (1:400; Zymed, South San Francisco, CA) and anti-ubiquitin (1:500; Dako).
Determination of cytochrome c distribution and caspase-3 activation used immunofluorescence with the nuclei stained with Bisbenzamide. Cytochrome c was detected using a mouse monoclonal antibody (1:500; PharMingen, San Diego, CA). Active caspase-3 was detected using a polyclonal rabbit antibody (1:500; Cell Signaling, Beverly, MA). The fluorescent products were imaged with a Zeiss laser scanning microscope.
RT-PCR and Real-time PCR were used with primers for b-actin forward and reverse and a-synuclein forward and reverse. Primers were designed by Primer Express (Applied Biosystems)
For control and rotenone-treated cells, glutathione levels were assayed using a glutathione assay kit (Cayman Chemical Co.). Protein carbonyls were assayed with the Oxyblot protein oxidation detection kit(Intergen)which uses a fluorescent assay with anti-8-hydroxydeoxyguanosine (8-oxo-dG) antibodies (Trevigen, Gaithersburg, MD) to measure oxidative DNA damage. Apotosis was measured with Apoptag Plus fluorescein in-situ apoptosis detection kit (Intergen)and the cell death was measured with Sytox green dye (Molecular probes).
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