Whole blood was extracted from mice or healthy human donors and prepared on the day of use. RBCs were separated from plasma by centrifuging 1 ml of whole blood at 300g at 20°C for 1.5 min. The supernatant was removed by aspiration. The packed RBCs were resuspended and washed three times in PBS buffer. The RBCs were then diluted with a PBS solution (3%, v/v). Note that for all mRBCs, PBS buffer was prepared as follows: 152 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.47 KH2PO4, and 10 mM glucose (pH 7.4) (osmolality, 340 mOsm/kg). For hRBCs, PBS buffer was used as purchased from Thermo Fisher Scientific (pH 7.2) (osmolality, 280 to 320 mOsm/kg; catalog no. 20012027). PEP (phosphoenolpyruvate) and Pi (sodium phosphate) solutions were prepared in PBS as follows: 50 mM PEP, 28.8 mM Mannitol, 50 mM glucose, 20 mM NaCl, and 1 mM adenine for PEP solution; and 50 mM Pi, 28.8 mM Mannitol, 50 mM glucose, 20 mM NaCl, and 1 mM adenine for Pi solution. pH of the solutions was adjusted to 6.0. Packed RBCs were suspended in either PEP or Pi solution, incubated for 1 hour at 37°C, and then washed twice with PBS. To determine the concentration of 2,3-DPG in RBCs, ultraviolet testing was conducted following the manufacturing protocol of Sigma Kit (catalog no. 10148334001, Sigma-Aldrich). In pervanadate treatment, packed RBCs were suspended in PBS with 0.5 mM sodium orthovanadate and 150 mM H2O2. After incubation at 37°C for 30 min, RBCs were washed three times using PBS and resuspended in a fresh PBS with glucose for 180 min to obtain 100% phosphorylation of band 3 and ankyrin.

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