Large-volume phlebotomy fails to induce a marked reduction in liver nonheme iron concentration in mice with hepatocyte-targeted Ncoa4 knockdown. (A) Experimental design. Eight-week-old C57BL/6N mice raised on a 45 ppm iron-replete diet underwent a single subcutaneous (SC) injection of saline vehicle, GalNAc-Luc-siRNA (3 mg/kg), or GalNAc-Ncoa4-siRNA (3 mg/kg) 7 days before phlebotomy. On experimental day 0, mice in each treatment group underwent a single 500-μL phlebotomy, which was immediately followed by (1) intraperitoneal volume replacement, (2) a second SC injection matching the initial treatment (ie, a booster dose), and (3) transfer to a 2 to 6 ppm iron-deficient diet (ID). (B-H) Murine parameters assessed 7 days after phlebotomy. To provide an experimental baseline for comparison, data from 8-week-old nonphlebotomized mice that were maintained on the iron-replete diet and did not receive any injections are also presented (Baseline group). (B) Hepatic Ncoa4 mRNA expression relative to Actb. The mean mRNA ratio obtained from livers of the baseline group was normalized to 1. (C) Immunoblotting analyses of NCOA4, TFRC, and β-actin in liver protein lysates. The black arrowhead denotes a 70-kDa band corresponding to the expected molecular weight of NCOA4. (D) Hgb level. (E) RBC. (F) Transferrin saturation. (G) Liver nonheme iron concentration. (H) Immunoblotting analyses of FTL, FTH, and β-actin in liver protein lysates. n = 4-7 per group. For all bar graphs, data represent mean ± SD. In panels C and H, numbers on the right indicate the position of molecular weight markers in kiloDaltons. ****P < .0001 by 1-way ANOVA with Tukey’s post hoc test.