O2-dependent cytosol/membrane migration of GAPDH is absent in SSRBCs. Confocal immunofluorescent imaging was used to observe GAPDH migration in intact RBCs during O2 loading/unloading. Washed RBCs were either fully oxygenated (HbO2% > 95%) or deoxygenated (HbO2% < 20%), fixed under appropriate gas tensions, probed for GAPDH (green) or cdB3 (yellow; see “Methods”), and confocal images were obtained (FluoViewFV1000 Olympus confocal microscope, PlanApo 60×/1.40 oil objective; Flouview acquisition software; imaging medium Prolong Gold). Membrane-based location of cdB3 is easily visualized in all images from both SS and normal RBCs. SSRBCs fail to demonstrate normal O2 content–dependent migration of GAPDH from cytosol to membrane, as demonstrated in normal RBCs on oxygenation.
