The increased septin content in spherocytosis is largely restored upon splenectomy and septin-2 associates with the RBC surface. RBCs from patients, either spl (filled circles or semifilled circles for intrafamily comparison) or nonspl (open circles), and RBCs from CTLs were compared for septin content (A-F) and membrane distribution (G). Statistics are indicated above the patient cohorts for the comparison with CTL values and above a horizontal line for comparison between the 2 patient cohorts, respectively. (A-F) Septin-2 and -7 ghost membrane association determined by western blotting. Representative western blots comparing different patients with healthy donors (A) and P13 before and after splenectomy (D). (B-C,E-F) Quantification: data were first expressed as ratio to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (loading control), then as ratio of P to CTL (Kruskal-Wallis test in panels B-C; means ± standard deviation of 1 experiment in panels E-F). (G) Septin-2 immunolabeling. Scale bar, 5 μm. RBCs were spread onto PLL-coated coverslips, fixed/permeabilized in ice-cold methanol and Triton X-100, and immunolabeled for septin-2. K562 cells were used as positive controls. Representative images of 2 independent experiments. (H-I) Correlation between septins and reticulocyte count presented in supplemental Figure 1E or Transferrin receptor (TfR) content presented at Figure 6K. Linear regressions were indicated only if r2 > 0.5.
