Vdac1 sc 8828

Whole-cell protein extracts were obtained by lysing cells with Laemmli sample buffer. Protein concentration was measured with the Micro BCA Protein Assay Reagent kit (Pierce, Rockford, Illinois, USA). 20–40 µg of protein extracts were subjected to reducing conditions, loaded onto a polyacrylamide gel, and then transferred to Immobilon-P membranes from Millipore (Billerica, Massachusetts, USA). One hour after blocking the nonspecific binding sites with 5% (w/v) non-fat milk in Tris-buffered saline with Tween® 20, membranes were incubated overnight at 4 °C with the following specific primary antibodies: β-actin (AC-15, Sigma-Aldrich), PHB1 (sc-28259, Santa Cruz, Dallas, TX, USA), PHB2/REA (07–234, Millipore), VDAC1 (sc-8828, Santa Cruz), HSP60 (4870 S, Cell Signaling, Danvers, MA, USA), GRP75 (sc-13967, Santa Cruz), MTCO2 (ab110258, Abcam, Cambridge, UK), PINK1 (6946, Cell Signaling). Antibody binding was detected using a secondary antibody conjugated to horseradish peroxidase, and the enhanced chemiluminescence detection system (Amersham, Little Chalfont, UK). Quantification of band intensities was conducted using Multi Gauge V3.0 software (FujiFilm Corporation). The relative density of each protein and condition was referred to the internal normalization control.