Cptc top1mt 3

Cells were seeded at 1.5 × 10⁴ cells over 12 mm glass coverslips (no. 1.5) in 24-well plates and incubated for 2 days. Subsequently, cells were fixed with 4% paraformaldehyde. Mitochondrial networks were labeled with a primary antibody against TOMM20 (Sigma-Aldrich, HPA011562) used at 1:1000, while TOP1MT was labeled with anti-TOP1MT-3 (Developmental Studies Hybridoma Bank, CPTC-TOP1MT-3; 1:200). Appropriate Alexa fluor–conjugated secondary antibodies (Thermo Fisher Scientific) were used at 1:1000.

Cell pellets were collected after trypsinization, washed with 1× PBS, and lysed with RIPA buffer (Thermo Scientific, 89900) containing protease inhibitors. Twenty to fifty micrograms of total cell lysates were resolved on SDS-PAGE gels and transferred onto PVDF membranes. Blots were probed with the following antibodies at the indicated dilutions; OxPhos antibody cocktail (Abcam, ab110411; 1:500 dilution), anti-Actin (Sigma, A5316; 1:1000), anti-NDUFB6 (Abcam, ab110244; 1:1000), anti-MTCO2 (Abcam, ab110258; 1:1000), anti-GAPDH (Millipore Sigma, ABS16; 1:1000), anti-VDAC1 (Abcam, ab14734; 1:1000), and anti-TOP1MT-3 (Developmental Studies Hybridoma Bank, CPTC-TOP1MT-3; 1:200). Appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies were used at 1:3000 as follows: goat anti rabbit IgG, HRP-linked Antibody (Cell Signaling Technology, 7074S), or goat anti-mouse IgG-HRP (Santa Cruz Biotechnology, sc-2055). Blots were incubated with Clarity ECL substrate (Biorad, 1705061) and imaged using an Amersham Imager AI600.