Cells were treated as described previously and harvested by cold Radioimmunoprecipitation Assay (RIPA) lysis buffer containing protease inhibitors and phosphatase inhibitors. After centrifugation at 12,000g for 20 min at 4°C, the protein concentration was assayed using the Bicinchoninic acid (BCA) assay. Equal amounts of protein (30 µg) were separated by 10-12% sodium dodecyl sulfate (SDS)-Polyacrylamide gel electrophoresis (PAGE) and transferred onto 0.45-µm polyvinylidene uoride (PVDF) membranes (Millipore, CA, USA). After the membranes were blocked with 5% fat-free dry milk in Tris-buffered saline containing Tween-20 (TBST), they were incubated with anti-Tom20, anti-Pink1, anti-Parkin, anti-LC3, anti-p62, anti-Bcl-2, anti-Bax, anti-cleaved-caspase-3, anti-ATP5A1 and anti-UQCRC1 at 4°C overnight. Membranes were washed three times and incubated with the HRP-conjugated secondary antibodies.
Protein bands were visualized by using ECL reagents and performed with the ChemiDicTM XRS + Imaging System (Hercules, CA, USA). Densitometry analysis of each band was quanti ed using Image Lab software and normalized against those of β-actin protein in each sample.
Protein bands were visualized by using ECL reagents and performed with the ChemiDicTM XRS + Imaging System (Hercules, CA, USA). Densitometry analysis of each band was quanti ed using Image Lab software and normalized against those of β-actin protein in each sample.