Ndufa4l2

Western blotting was performed as previously described.^{8 (link)} Primary antibodies were used (1:1,000 in 5% milk): NDUFA4L2 (Abcam ab74138, rabbit polyclonal), NDUFA4L2 (Proteintech 66050-1-Ig, mouse monoclonal), FLAG (GenScript, A00187S, mouse monoclonal). Secondary antibodies: anti-Rabbit IgG (Jackson, 711–135-052, 1:10,000 diluted in 5% milk), anti-Mouse IgG (Jackson, 715–035-150, 1:10,000 diluted in 5% milk).

Primary tumor cells were seeded at a density of 2 × 10⁵ on glass coverslips and left to adhere overnight at 37 °C in 5% CO₂. These preparations were stained for NDUFA4L2 (Proteintech, Chicago, IL, USA) and MitoSOX red (Molecular Probes, Life Technologies, Monza, Italy). The expression and localization of proteins was evaluated by indirect immunofluorescence and confocal microscopy analysis. In particular, the cells were fixed using ice-cold 4% paraformaldehyde for 10 min at room temperature. Then, the preparations were blocked with 1% BSA in PBS for 1 h at room temperature and incubated overnight at 4 °C with a primary antibody against NDUFA4L2 (1:25 in blocking), followed by incubation for 1 h at 37 °C with the secondary antibody goat anti-rabbit IgG FITC (1:200; Novus Biologicals). To study mitochondrial superoxide generation, live cells were stained with 5 μM MitoSOX red for 10 min at 37 °C. All preparations were counterstained with TO-PRO-3 (Molecular Probes). Negative controls were performed by omitting the primary antibodies. Specific fluorescence was acquired by a Leica TCS SP2 (Leica, Wetzlar, Germany) confocal laser-scanning microscope using a ×63 objective lens.