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Then the membrane was incubated with rabbit anti-Glutathione Peroxidase 4/GPX4 antigen affinity purified polyclonal antibody (A02059-1) at 0.5 g/mL overnight at 4C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody (Catalog # BA1054) at a dilution of 1:5000 for 1.5 hour at RT