Loss of endosomal targeting of the Steap3^Y288H reduces ferrireductase activity and the uptake of iron and copper. (A) Ferrireductase activity in whole blood preparations: incubations were carried out for 30 minutes and normalized to TCA RNA (n = 11 wild-type; n = 8 mutant). (B) Serum iron (n = 11 WT; n = 9 mutant); (C) liver hepcidin, expressed as cycle threshold (Ct) (n = 8 WT; n = 12 mutant); (D) TIBC (n = 8 WT; n = 6 mutant); and (E) transferrin saturation (n = 8 WT; n = 6 mutant). (F) Liver iron in male and female WT (n = 5 or 7) and mutant mice (n = 3 or 6). (G) Serum and (H) liver copper in WT (n = 12) and mutant mice (n = 9). Graphs show means (columns) and SDs of the mean (bars). Statistical analysis was performed using unpaired t test to WT controls: *P = .01 to .05; **P = .001 to .01; ***P = < .001. (I) Localization of FLAG-tagged wild-type Steap3 (Steap3^WT) and Steap3^Y288H in HEK-293T cells in images taken at ×40 and ×100 magnification. Images were obtained using a Zeiss Axioplan 2 upright microscope and a 63×/1.4 NA oil-immersion objective and Zeiss Meta confocal imaging software (Zeiss, Thornwood, NY). Bars = 10 μm.