Band 3 and ABCB6 overexpression in plasma membrane protein of RBCs from patients with DHS and PIEZO1–band 3 colocalization. (A) Left: representative immunoblot of RBC membrane proteins showing band 3 and ABCB6 protein expression normalized to β-actin in the patients DHS1, DHS2, DHS3, DHS4, DHS5, and DHS6 compared with HCs (2 pools of HCs, each of n = 3). Right: densitometric analysis of 3 representative western blotting is shown for band 3 and ABCB6 proteins. Data are presented as mean ± standard deviation; ∗P < .05; ∗∗P < .01 (Student t test). (B) Representative confocal imaging by ZEISS LSM 980 Airyscan 2 of HC RBCs treated with vehicle (upper panel) compared with HC RBCs treated with 2.5 μM Yoda1 (10 minutes) (middle panel) and HC RBCs treated with 2.5 μM Yoda1 (20 minutes) (lower panel). Rabbit anti-PIEZO1 antibody is shown in green, mouse anti–band 3 is shown in red. The overlapping of both signals (MERGE) is shown on the right (yellow). (C) Quantification by Pearson correlation of PIEZO1–band 3 colocalization in untreated HC RBCs, HC RBCs + 2.5 μM Yoda1 (10 minutes), and HC RBCs + 2.5 μM Yoda1 (20 minutes) is shown. Data shown are the means of 12 independent acquisitions ± standard error of the mean; P < .05 for trend (ANOVA test), ∗P < .05 (post hoc correction by Tukey multiple comparisons test).