Polyclonal anti cleaved active caspase 3 antiserum

Cytosolic or mitochondrial fraction of SH-SY5Y dopaminergic cells was prepared as described above. Subsequently, 30 μg of cytosolic or mitochondrial protein was separated on 12% SDS-polyacrylamide gel and transferred to PVDF membrane. Then, the membrane was incubated at 4 °C overnight with one of the following diluted primary antibodies: (1) Polyclonal anti-cleaved active caspase 9 antibody (Cat.9508, Cell Signaling Technology). (2) Polyclonal anti-cleaved active caspase 3 antiserum (Cat.9662, Cell Signaling Technology). (3) Polyclonal anti-Bax antibody (Cat.2772, Cell Signaling Technology). (4) Polyclonal anti-cytochrome c antiserum (Cat. ab13575, Abcam). (5) Monoclonal anti-TOM20 antibody (Cat.42406, Cell Signaling Technology). (6) Polyclonal anti-Atg7 antiserum (Cat.2631, Cell Signaling Technology). (7) Monoclonal anti-p62 antibody (Cat.8025, Cell Signaling Technology). (8) Polyclonal anti-LC3-I/II antiserum (Cat.4108, Cell Signaling Technology). After the wash, the membrane was incubated with anti-rabbit secondary antibody conjugated with horseradish peroxidase (HRP). Then, immunoreactive proteins were visualized by using an ECL kit. Relative protein expressions were normalized with the level of β-actin using a densitometer (Molecular Dynamics Model 375A).

Protein samples were prepared by homogenization of SH-SY5Y cells or SN neurons in SDS sample buffer. Subsequently, protein lysate or the immunocomplex was separated on 8 or 10% SDS-polyacrylamide gel and transferred to PVDF membrane. Then, the membrane was incubated at 4 °C overnight with one of the following diluted primary antibodies: (1) Anti-FLAG monoclonal antiserum (Sigma-Aldrich). (2) Polyclonal anti-Complex IV antibody (Sigma-Aldrich). (3) Polyclonal anti-cleaved active caspase 9 antibody (Cell Signaling Technology). (4) Polyclonal anti-cleaved active caspase 3 antiserum (Cell Signaling Technology). (5) Polyclonal anti-Bax antibody (Cell Signaling Technology). (6) Polyclonal anti-cytochrome c antiserum (Abcam). After the washes, the membranes were incubated with horse anti-mouse, anti-donkey or anti-rabbit horseradish peroxidase (HRP) linked secondary antibodies. Then, immunoreactive proteins were visualized by using an enhanced chemiluminescence kit (GE Biosciences). To confirm equal amount of protein sample loading, membranes were stripped and reblotted with monoclonal anti-actin antibody (Chemicon). Gel bands were quantified by a densitometer (Molecular Dynamics Model 375A) and normalized by reprobing the same blot for the actin signals.