The extracted total cell lysate and mitochondrial proteins of H5N1 and H9N2 virus-infected A549 cells were quantified with a BCA kit (Sangon Biotech, Shanghai, China). Mitochondrial proteins (40 µg) and total cell lysate (40 µg) were separated by 12% SDS-PAGE and transferred to nitrocellulose membranes (BBI Life Sciences). After blocking with 5% (w/v) skim milk in TBST (50 mM Tris [pH 8.0], 150 mM NaCl, 0.1% [v/v] Tween-20) for 1 h at 37 °C, membranes were incubated separately overnight at 4 °C with rabbit monoclonal or polyclonal antibodies against ECHS1 (ab170108), MDH2 (ab181873) (Abcam), ATP5F1 (15999-1-ap), HSPA1L (13970-1-ap), BAX (50599-2-Ig), and Caspase 3 (66470-2-Ig) (Proteintech). After three washes with TBST, membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG or HRP-conjugated goat anti-mouse IgG (used at a 1:5000 dilution, Proteintech) for 1 h at room temperature and were then washed three times with TBST. The immunoreactive protein bands were detected using enhanced chemiluminescence reagent (ECL; Advansta, CA, USA), with TOM40 (18409-1-ap) (Proteintech) and β-actin (66009-1-Ig) (Proteintech) as the loading controls.

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