GalNAc-Ncoa4-siRNA treatment produces durable Ncoa4 knockdown in mouse liver and raises hepatic nonheme iron concentration. Shown are analyses of parameters assessed in mouse livers harvested 1, 2, 3, or 4 weeks after a single injection of vehicle, GalNAc-Luc-siRNA (3 mg/kg), or GalNAc-Ncoa4-siRNA (3 mg/kg). (A) Hepatic Ncoa4 mRNA expression relative to Actb. (B) Immunoblotting analyses of NCOA4, TFRC, and β-actin in liver protein lysates. Black arrowhead denotes a 70-kDa band corresponding to the expected molecular weight of NCOA4. (C) Liver nonheme iron concentration. (D) Immunoblotting analyses of FTL, FTH, and β-actin in liver protein lysates. The mean band density of FTL or FTH relative to β-actin from livers harvested 4 weeks after vehicle injection was normalized to 1. (E) Hepatic Tfrc mRNA expression relative to Actb. For panels A and E, the mean mRNA ratio obtained from livers harvested 1 week after vehicle injection was normalized to 1. For panels B and D, numbers on the right indicate the position of molecular weight markers in kiloDaltons. n = 5 per group. For all bar graphs, data represent mean ± SD. *P < .05, **P < .01, ***P < .001, and ****P < .0001 by 2-way ANOVA (panels A, C, E) or 1-way ANOVA (panel D) with Tukey’s post hoc test.