Enhanced chemiluminescence analysis kit

Stable H9c2 cells, mouse heart, and isolated mitochondrial fractions were lysed with RIPA buffer, and protein content was measured using the Bradford assay. Cell and tissue homogenate protein was separated by 1‐dimensional gel electrophoresis. After transfer to a polyvinylidene difluoride membrane, the membrane was incubated with antibodies that recognize proteins such as mt‐COX1 (Abcam, Cambridge, MA), mt‐COX2 (Life Technologies, Carlsbad, CA), mt‐COX3 (Life Technologies, Carlsbad, CA), COX 5A (Abcam, Cambridge, MA), COX 5B (Abcam, Cambridge, MA), COX VIIa (Abcam, Cambridge, MA), pAkt at Ser 473 (Cell Signaling, Danvers, MA), Akt (Cell Signaling, Danvers, MA), α‐tubulin (Abcam, Cambridge, MA), and VDAC (Abcam, Cambridge, MA) in Tris‐buffered saline (pH 7.4) with 1% TWEEN 20 (TBS‐T) and 5% BSA or nonfat dry milk at 4°C overnight. Membranes were incubated with the appropriate secondary antibody conjugated to horseradish peroxidase IgG in TBS‐T with 5% nonfat dry milk for 1 hour at room temperature. Immunoreactive protein was visualized using an enhanced chemiluminescence analysis kit (EMD Millipore, Temecula, CA).