Ab224565

Anti-Flag was purchased from Sigma Aldrich (F1804, Sigma Aldrich, Saint Louis, MO, USA). Voltage-dependent anion channel (VDAC) (D73D12) antibody was purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against Mic60 (ab137057), Mic10 (ab84969), CHCHD3 (ab224565), translocase of the outer membrane 20 (Tom20) (ab56783), heat shock protein 60 (Hsp60) (ab46798), glyceraldehydes-3-phosphate dehydrogenase (ab181602), β-actin (ab8226), and tyrosine hydroxylase (TH) (ab112) were from Abcam (Cambridge, UK). The anti-mouse and anti-rabbit secondary antibodies (1:1000) were purchased from Cell Signaling Technology. MitoTracker Deep Red was obtained from Life Technologies (Carlsbad, CA, USA) (M22426). 1-methyl-4-phenyl-1,2,3,6-tetrahydropyri-dine (MPTP) hydrochloride and methyl-4-phenylpyridi-nium (MPP⁺) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

To detect ectopically‐expressed MIC19, a HA tag was inserted directly after the myristoylation motif (a.a. 1–14) of MIC19. We have confirmed that the HA tag does not affect mitochondrial targeting of MIC19. U2OS cells stably expressing HA‐MIC19 were grown on coverslips coated with poly‐l‐lysine (Sigma‐Aldrich). Mitochondrial staining was performed by incubating cells with growth medium containing 25 nM of MitoTracker probe (Thermo Fisher) for 45 min at 37°C. After rinses with PBS, cells were fixed in 4% paraformaldehyde for 15 min at room temperature, followed by three washes in 0.2% Triton X‐100/PBS. The coverslips were blocked with blocking buffer (2% BSA in 0.2% Triton X‐100/PBS) for 1 h and then incubated overnight with a MIC19 primary antibody (Abcam, ab224565) (1:100 dilution in blocking buffer) at 4°C. Following three washes in 0.2% Triton X‐100/PBS, cells were incubated with Alexa Fluor‐conjugated secondary antibody (Thermo Fisher, A27034) (1:100 dilution in blocking buffer) for 1 h at room temperature in the dark. The coverslips were mounted on glass slides using Fluoroshield DAPI mounting medium (Sigma‐Aldrich). Images were captured using a scanning laser confocal microscope (ZEISS LSM710). For quantification analysis, all images were acquired using the same device parameters.