Effects of Steap3 nonsynonymous polymorphisms between B6 and FVB strains. Western blot of CHO cell lysates from cells transiently expressing recombinant Steap3 variants (A) or RBC ghosts (C). Blots were probed by using an anti-Steap3 antibody; after development, membranes were stripped and re-probed with anti-actin as a control for loading and transfer. (B) Ferrireductase activity in transfected CHO cells as measured by using ferrozine as an indicator of conversion of Fe³⁺ to Fe²⁺. Data shown represent combined means from 5 independent experiments. Statistics were calculated by using 2-way ANOVA with a Bonferroni post hoc analysis. At the highest concentration of CHO cells tested (inset panel), ferrozine activity of the transfected B6 Steap3 variant was significantly lower than A350V (*P < .01) and A350V+N455S (***P < .0001) but not different from N455S by itself. (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), ferrozine activity of B6 was not different from B6.FVB-A350V. (E) 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.