The ANK1 content and the SPT membrane distribution are differentially affected by the mutation but not by splenectomy and inversely and slightly correlate together and with the RBC alteration score. RBCs from patients, either spl (filled circles or semifilled circles for intrafamily comparison) or nonspl (open circles), and RBCs from CTLs (dark blue dotted line, CTL ratio or % CTL) or a healthy spl adult donor (red dotted line) were compared for SPTB and ANK1 content (A-D) and membrane SPTB coverage and heterogeneity (E-I). 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. (A-D) SPTB and ANK1 membrane association evaluated by mass spectrometry on ghosts (2 independent experiments; see Figure 3 for volcano plots). Data are presented based on either mutation (A-B) or splenectomy (C-D). (E-I) SPT occupancy and heterogeneity. RBCs were spread onto PLL-coated coverslips and SPT was immunolabeled and observed via confocal microscopy. (E) Representative images (filled arrowhead, SPT-enriched patch; open arrowhead, SPT-enriched vesicle). Scale bar, 5 μm. In panels F-I, SPT occupancy was normalized to RBC membrane area (means of 2-7 independent experiments per patient; Kruskal-Wallis tests followed by Dunn post hoc). Data are presented based on either mutation (F-G) or splenectomy (H-I). (J-K) Relations between the ANK1 levels and the SPT coverage and variance. (L-M) Relations between the RBC alteration score from Figure 1N and the ANK1 levels or the SPT variance.