Anti tom20 antibody

Cells were collected and seeded onto glass coverslips processed for immunofluorescence. The glass coverslips were washed twice with cold PBS for 5 min, fixed with 4% paraformaldehyde for 20 min and incubated with 0.2% Triton X-100 for 5 min at 4 °C. After incubation, the cells were blocked with PBS containing 3% BSA for 1 h and incubated with anti-HK2 antibody (1:100, abclonal), anti-p-AKT antibody (1:100, abclonal) as well as anti-TOM20 antibody (1:400, abcam) overnight. After being washed twice with cold PBS for 10 min, the cells were stained with FITC-conjugated Goat Anti-Rabbit and PE-conjugated Goat Anti-Mouse IgG second antibody (1:500, abcam) for 1 h. The images were captured with a confocal microscope (Olympus FV1000, Olympus Corp., Tokyo, Japan).

Immunofluorescence experiments were performed as detailed previously (36 (link),37 (link)). HeLa cells were cultured on cover glass slips (Thermo Fisher), fixed in 4% formaldehyde for 15 min, permeabilized with 0.2% Triton X-100, blocked with 5% Fetal Bovine Serum (FBS) for 1h, and immunestained with an anti-TOM20 antibody (Abcam) and anti-FLAG antibody (Abcam) overnight at 4°C. The cells were then incubated with Alex Fluor 594 goat anti-mouse IgG (H&L) and Alex Fluor 488 goat anti-rabbit IgG (H&L) (Thermo Fisher), stained with 4′,6-daimidino-2-phenylindole (DAPI) (Invitrogen) for 15 minutes, and mounted with Fluoromount (Sigma-Aldrich). Cells were examined using a confocal fluorescence microscope (Olympus Fluoview FV1000, Japan) with three lasers (Ex/Em = 550/570, 492/520 and 358/461 nm).