Levels and effects of Steap3 expression on RBCs. (A,C-D). Ferrireductase activity in RBCs as measured by using ferrozine as an indicator of conversion of Fe³⁺ to Fe²⁺. Data shown represent combined means from 3 independent experiments (n = 2-4 mice per group, per experiment). Statistics were calculated by using 2-way ANOVA with a Bonferroni post hoc analysis. At the highest concentration of RBCs tested (inset panel in each graph), ferrozine activity of B6 was significantly lower than all other samples (P < .0001) except Rec#8.1 for panel A; ferrozine activity of B6 was significantly lower than all other samples (**P < .005 for F1 and P < .0001 for FVB, B6.FVB-Chr1FVB/FVB) for panel C; and ferrozine activity of B6 was significantly lower than all other samples (P < .0001) for panel D. (B,E) Western blot of RBC ghosts using an anti-Steap3 antibody. After development, membranes were stripped and re-probed with anti-Actin as a control for loading and transfer. (F) Twenty-four–hour recoveries of RBCs stored for 7 days. These data represent combined means and SD from 3 independent experiments (experiment 1 data are shown as a solid circle, experiment 2 data are shown as a hollow circle, and experiment 3 data are shown as an X). Statistics were calculated by using 1-way ANOVA with a Bonferroni post hoc analysis. A significant difference from B6 is indicated by ***P < .0001.