Western Blot Analysis of Cellular Proteins

Proteins (60 μg) from all sample groups were processed as previously described [92 (link)]. Proteins were separated on SDS-PAGE and subsequently transferred to nitrocellulose sheets using a semidry blotting apparatus. Sheets were saturated for 60 min at 37 °C in blocking buffer (1xTBS, 5% milk, 0.05% Tween-20), then incubated overnight at 4 °C in blocking buffer containing primary antibodies to SOD1 (1:100, Abcam), Caspase 1 (1:100, abcam), BCL2 (1:100, Santa Cruz Biotechnology, Dallas, Texas, USA), BAX (1:100, Cell Signaling Technology), LC3A/B (1:100, Cell Signaling Technology) and β-actin (1:1000, Santa Cruz Biotechnology). After four washes in TBS containing 0.1% Tween-20, samples were incubated for 30 min at room temperature with peroxidase-conjugated secondary antibody diluted 1:1000 in 1× TBS, 5% milk, 0.05% Tween-20. Bands were visualized by the ECL method. The level of recovered protein was measured using the Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s instructions.