Effects of carboxyeosin on intracellular calcium levels in HNF1A^+/+and HNF1A^−/−RBCs. (A) Immunoblot analysis showing PMCA, band 3, glycophorin A, and β-actin protein expression in RBC ghosts prepared from HNF1A^+/+ and HNF1A^−/− mice (n = 3 each). (B) Representative FACS plots of intracellular calcium kinetics measured in Fluo-4–loaded HNF1A^+/+ and HNF1A^−/− RBCs under basal conditions and after subsequent ionomycin treatment (i). Flow cytometric determination of intracellular Ca²⁺ changes over time as measured by the emission of Fluo-4–loaded HNF1A^+/+ (n = 4) and HNF1A^−/− RBCs (n = 4) under basal conditions (set as t = 0) and after subsequent ionomycin treatment (t = 35 seconds and t = 147 seconds) (ii). ***P < .001 for untreated HNF1A^+/+ vs treated HNF1A^+/+. §§§P < .001 for t = 35 seconds vs t = 147 seconds HNF1A^+/+. *P < .05 and **P < .01 for untreated vs treated HNF1A^−/−. (C) FACS plots of intracellular calcium kinetics measured in Fura Red–loaded HNF1A^+/+ and HNF1A^−/− RBCs as emission ratio (405 nm/488 nm) and fluorescence emission intensities at each of the 2 component wavelengths (FL10 excited by 405 nm and FL4 excited by 488 nm) under basal conditions and after ionomycin. (D) Flow cytometric determination of intracellular Ca²⁺ changes over time as measured by the ratio alteration of Fura Red–loaded HNF1A^+/+ (n = 3) (i) and HNF1A^−/− (n = 3) (ii) RBCs after incubation for 30 minutes (set as 100%) with the PMCA inhibitor carboxyeosin (CE) (10 μM) or vehicle (DMSO) under basal conditions (set as t = 0) and after subsequent ionomycin treatment. *P < .05 for untreated HNF1A^+/+ vs treated HNF1A^+/+ RBCs. §P < .05 and §§§P < .001 for ionomycin-treated RBCs (t = 35 seconds vs every later time point). #P < .05 and ###P < .001 for ionomycin- and CE-treated RBCs (t = 35 seconds vs every later time point).