Quantitative Analysis of Mitochondrial Proteins

Different quantities (2, 4, 8, and 8 μg) of crude mitochondrial membrane protein extracts from the developing kernels at 11 DAP were separated by 12.5% SDS-PAGE. After transferring to a nitrocellulose membrane (0.45 mm; Millipore), the membrane was incubated with primary antibodies using polyclonal rabbit antibodies against Nad9, Cyt_C, Cyt_C1, Cox2, ATPase, and AOX as described previously (Liu et al., 2020a (link)). The HRP-conjugated goat anti-rabbit antibody (Abcam) was used as the secondary antibody. Signals were visualized on X-ray films (Kodak, Tokyo, Japan) using the ECL reagents (Pierce, Thermo Fisher Scientific, Waltham, MA, United States) according to the manufacturer’s instructions.