Mouse monoclonal antibody to ndufa9

Cultured cells were collected and lysed in buffer composed of a complete protease inhibitor. Protein contents of cell lysates were determined using the Pierce BCA Protein Assay Kit (Thermo Scientific). Lysates equivalent to 20 µg of protein were electrophoresed on 15% SDS-PAGE gels and then transferred onto PVDF membranes (Millipore, USA). Membranes were blocked for 90 min with 10% nonfat dry milk in PBS containing 0.1% Tween-20 (PBST) at room temperature (25℃) and probed with the following primary antibodies overnight at 4℃: mouse monoclonal antibody to ISCU (1:500, Santa Cruz Biotechnology, USA), mouse monoclonal antibody to NDUFA9 (1:1000, Abcam, UK), rabbit monoclonal antibody to SIRT3 (1:1000, CST, USA), mouse monoclonal antibody to β-actin (1:2000, Proteintech, USA). Then, the membranes were washed with PBST and probed with a secondary horseradish peroxidase-conjugated anti-mouse or antirabbit IgG antibody (1:1000, Beyotime, China) for 1 h at 25℃. The immunoreactive bands were visualized using the SuperSignal West Pico kit (Thermo Scientific). Band intensity was quantified by Image J.