Iron binding to different lobes of Tf differentially affects erythropoietin levels despite similar erythroid signaling and iron availability for heme synthesis. (A) Serum Epo concentrations were significantly elevated in Tf^N-bl/N-bl mice relative to both Tf^C-bl/C-bl and WT mice (n = 5 mice per sex for each mutant strain and n = 10 mice per sex for WT). (B) Tf^C-bl/C-bl mice showed significant increases in GypA messenger RNA (mRNA) levels relative to control mice (n = 4-7 mice per sex for WT and n = 3 mice per sex for mutant mice). (C) Erythroferrone (Erfe) mRNA levels were significantly higher in both Tf^N-bl/N-bl and Tf^C-bl/C-bl mice compared with controls (n = 4-7 mice per sex for WT and n = 3 mice per sex for mutant mice). (D) Upon normalization to GypA, Erfe expression was significantly higher in the Tf^C-bl/C-bl mice relative to controls (n = 4-7 mice per sex for WT and n = 3 mice per sex for mutant mice). (E) Tf^N-bl/N-bl and Tf^C-bl/C-bl mice demonstrated elevated zinc protoporphyrin (ZnPP) concentrations per mole of heme (n = 9-7 mice per sex for WT and n = 5-8 mice per sex for mutant mice). Data are presented as Tukey box and whisker plots, with statistical significance analyzed by 1-way ANOVA. *P ≤ .05, ***P ≤ .001, ****P ≤ .0001.