Fig 2: Loss of surface area as reticulocytes mature exceeds the loss of skeletal proteins. (a, b) HEMA analysis of volume and surface area for CD71+, CD71- reticulocytes and mature RBCs from the same sample. Unpaired T-test: *** = p = 0.001. (c) A schematic highlighting approximate protein positions within the junctional complexes. (d-i) Fluorescence intensity from sheared membranes stained with antisera against different membrane proteins. Representative images (partial fields) are shown for each protein, along with data for mean intensity values per full 60X field for each cell type. CD71+ cells are labelled and plotted in green, with mature cells labelled by CR1 plotted in red. CD71- cells are unlabelled and are plotted in blue, with membrane protein staining in greyscale. Scale bar = 5 µm. (j) Ratios of fluorescence intensities for CD71 + and CD71- reticulocytes relative to mature RBCs (means and standard deviations are shown, minimum of 10 image fields per repeat). Unpaired T-test: * = p = 0.05; ** = p = 0.01; *** = p = 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig 3: Moderate shrinking of the physical skeletal meshwork is evident as reticulocytes mature. (a–c) SEM of whole cells and representative images of sheared membranes (d-f) for CD71+ (a,d) and CD71- (b,e) reticulocytes as well as mature RBCs (c,f) separated from peripheral blood and bound to PHA-E coated surfaces. Scale bars = 5 μm (a–c) and 500 nm (d–f). (g, h) Skan analysis was performed on sheared membrane networks from 9 independent experiments (3 cord blood, 3 peripheral blood, 3 magnetically enriched peripheral blood samples) with data for CD71+ (g) and CD71− (h) reticulocytes expressed as ratios of values obtained from mature RBCs within the same sample.