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The following antibodies were used: anti-GPX4 (1:2000, Abcam, ab125066), anti-ACSL4 (1:1000, Abcam, ab155282), anti-TfR (1:1000, Invitrogen, H68.4), anti-FPN1 (1:1000, Eurogentec, Lege, Belgium) anti-ferritin (1:1000, Sigma, F5012), anti-phospho-NF-B p65 (1:1000, Cell Signalling Technology, 3039), anti-NF-B p65 (1:1000, Cell Signalling Technology, 8242), with anti-GAPDH (1:2000, Cell Signalling Technology, 2118) or anti--actin (1:2000, Sigma, A2066 and Abcam, ab49900) as loading control

Given the ancient origin of the Ca 2+ -based signaling, which evolutionary developed earlier than the mitochondrial apoptotic toolkit whose emergence cannot be dated back to the times earlier than eukaryogenesis and acquisition of an endosymbiotic mitochondrion, it can be assumed that the Ca 2+ toolkit takes over a key role in cell death signaling in terminally differentiated mitochondria-free erythrocytes

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