Anti ferritin

The Proximity ligation assay was performed using Duolink™ In Situ Red Starter Kit Mouse/Rabbit (CAT#DUO92101-1KT, Sigma-Aldrich) following the manufacturer’s protocol. Briefly, after permeabilization, cells were blocked against nonspecific binding with Duolink® Blocking Solution for 2 h at 37 °C. Anti-NCOA4 (CAT#sc-373739, Santa Cruz) and Anti-ferritin (CAT#4393, Cell Signaling Technology) were then incubated with cells in Duolink® Antibody Diluent overnight at 4 °C. After washed with wash buffer A, cells were incubated with Duolink® PLA Probe for 60 min at 37 °C. Cells were then incubated with the ligase and Duolink® Ligation buffer for 30 min at 37 °C. Next, cells were washed in wash buffer A for 10 min at room temperature (RT) and incubated with the polymerase and amplification buffer for 100 min at 37 °C. Finally, cells were washed in wash buffer B and 0.01× wash buffer B at RT. Then, cells were mounted with Duolink® In Situ Mounting Medium with DAPI and imaged with FV1000 confocal microscopy (Olympus).

Liver homogenates (20 µg) were electrophoresed on 12% SDS/polyacrylamide gels. Proteins were then transferred on to nitrocellulose membranes (0.2 µm pore size) (Bio-Rad Laboratories, Gladesville, NSW, Australia) using a Trans-blot Turbo (Bio-Rad) blotting system. The membrane was then blocked with 10% skim milk in Tris buffered saline with 0.1% Tween-20 (TBST) (Sigma–Aldrich)) and then incubated with anti-GAPDH (1:360000, Merck Millipore, Bayswater, Victoria, Australia) and anti-ferritin (1:5000, Cell Signaling, Danvers, Massachusetts) diluted in 10% skim milk overnight at 4°C. The membrane was washed thrice with TBST before being incubated with secondary anti-rabbit or anti-mouse IgG conjugated to horseradish peroxidase (1:1000) (65-6120, Invitrogen, Waltham, Massachusetts) diluted in 10% skim milk for 1 h at room temperature. The membrane was further washed before being incubated with chemiluminescent substrate (Lumina Forte; Merck Millipore) and imaged on a Chemidoc imaging system (Bio-Rad) for various timepoints. The blots were quantitated using ImageJ (National Institutes of Health, Bethesda, Maryland).