Western Blot Analysis of Mitochondrial Proteins

Cell lysates were collected and prepared as described previously [45 (link), 46 (link)]. Briefly, 661W cells were harvested and lysed in a Tris lysis buffer (in mM): 50 Tris, 1 EGTA, 150 NaCl, 1% Triton X-100, 1% β-mercaptoethanol, 50 NaF, and 1 Na₃VO₄, pH 7.5. Samples were separated on 10% sodium dodecyl sulfate-polyacrylamide gels by electrophoresis and transferred to nitrocellulose membranes. The primary antibodies used in this study were DRP1 (1 : 1000; Cell Signaling Technology, Danvers, MA, USA), MFN2 (1 : 1000 Abcam, Cambridge, MA, USA), MCU (1 : 1000 Abcam), and actin (loading control; 1 : 1000, Cell Signaling Technology). Blots were visualized using appropriate secondary antibodies (Cell Signaling Technology) at 1 : 1000 conjugated to horseradish peroxidase and an enhanced chemiluminescence (ECL) detection system (Pierce, Rockford, IL, USA). Band intensities were quantified by densitometry using Scion Image (NIH, Bethesda, MD, USA).