CD47 on experimentally senescent erythrocytes is recognized by SIRPα. (A) Experimental aging of erythrocytes in combination with serum treatment induces binding to CHO SIRPα cells. (B) CD47 staining with the conformation-independent antibody B6H12 and 2D3, an antibody that detects a conformation-dependent epitope of CD47 on experimentally aged erythrocytes, shows a conformational change of CD47 after experimental aging. (C) Experimental aging of erythrocytes results in binding of the TSP-1-derived peptide 4N1K. No binding was observed for the control peptide, 4NGG. Furthermore, no streptavidin-APC staining was observed for fresh erythrcoytes and oxidized erythrocytes when no peptide was added or when fresh erythrocytes were incubated with the 4NGG peptide (data not shown). (D) Aging of young erythrocytes in combination with 4N1K peptide incubation results in binding to CHO SIRPα cells. (E) Oxidation of CD47 beads inhibits binding to CHO SIRPα cells. The binding of oxidized CD47 beads could subsequently be induced in combination with the 4N1K peptide, but not with the control peptide, 4NGG. Data are representative of 3 experiments.