Immunoblotting Analysis of Mitochondrial Proteins

The protocol of extracting cells is as previously described [5 (link), 20 (link)]. The equal denatured protein underwent 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then was transferred to a nitrocellulose membrane by electrophoresis. Membranes were blocked with 5% fat-free milk and immunoblotted with diluted primary antibody: anti-phospho-JNK (1 : 400), anti-Sab (1 : 250), anti-p-Src (1 : 250), anti-c-Src (1 : 1000), anti-COX-IV (1 : 400), and anti-β-actin (1 : 5000) and then incubated at 4°C overnight, which was followed by exposure to horseradish peroxidase-conjugated secondary antibodies (typically 1 : 20,000 dilution) for 1 hour at room temperature. Transferred proteins were visualized by use of a SuperSignal detection kit (Thermo-Fisher Scientific, Waltham, MA, USA). The bands were analyzed with the Gel-Pro Image Analysis Software (Media Cybernetics, USA). The level of each mitochondrial protein was expressed as its density to the density of mitochondrial loading control COX-IV. To avoid the error in defferent batches, the desity ratio between interest protein and COX-IV in the H/R group was set as 1.