The cytosolic and mitochondrial fractions were isolated from cultured RGC-5 cells by differential centrifugation (Mitochondrial Isolation Kit; Pierce, Rockford, IL, used according to the manufacturer’s Dounce homogenizer procedure). For Western blot analysis, mitochondria were lysed with 2% CHAPS (3-[3-cholamidopropyl]dimethylammonio-2-hydroxy-1-propanesulfonate) in TBS for protein analysis with a DC protein assay (Bio-Rad, Hercules, CA). The cytosolic or mitochondrial fractions were mixed with SDS-PAGE sample buffer and boiled for 10 minutes. Equivalent amounts of protein (10 μg) for each sample were loaded onto 4% to 12% precast polyacrylamide gradient gels (Invitrogen). The proteins were electrotransferred to a nitrocellulose membrane in Tris-glycine-methanol transfer buffer. The membrane was blocked for 1 hour at room temperature in PBS containing 5% nonfat dry milk and 0.05% Tween-20 and then incubated for 15 hours at 4°C with primary antibodies: polyclonal rabbit anti-human Drp1 (dynamin-related protein-1) antibody (H-300, cat. no. sc-32898, 1:1000; Santa Cruz Biotechnology Inc., Santa Cruz, CA), monoclonal mouse anti-actin antibody (Ab-1, cat. no. CP01, 1:10,000; Calbiochem, La Jolla, CA) and polyclonal rabbit anti-VDAC (Porin) antibody (Ab-5, cat. no. PC548T, 1:1000, Calbiochem). The actin or VDAC antibodies were used to confirm similar cytosol or mitochondrial protein loading in each lane to the Western blot analysis for Drp-1, respectively. The membrane was then rinsed with 0.05% Tween-20 in PBS and incubated for 2 hours at room temperature with peroxidase-conjugated goat anti-rabbit IgG for Drp-1 and VDAC antibodies (1:2000; Bio-Rad) or goat anti-mouse IgM for actin antibody (1:2000; Calbiochem). The blots were developed with a chemiluminescence detection kit (ECL Plus; GE Healthcare Bio-Sciences, Piscataway, NJ), used according to the manufacturer’s recommendations. Images were analyzed by using a digital fluorescence imager (Storm 860; GE Healthcare Bio-Sciences). Band densities on the Western blot analysis were determined by computer (ImageQuant TL Analysis software; GE Healthcare Bio-Sciences).