HJV overexpression causes splenic iron retention in control animals. (A) SIC was increased in control animals after HJV overexpression (control PBS, n = 11; control AAV-HJV, n = 10; Alk2^fl/fl;Alb-Cre PBS, n = 7; Alk2^fl/fl;Alb-Cre AAV-HJV, n = 5; Alk3^fl/fl;Alb-Cre PBS, n = 6; and Alk3^fl/fl;Alb-Cre AAV-HJV, n = 6). (B) Iron retention in the spleen was confirmed by Prussian blue staining. (C-D) Splenic ferroportin protein levels were determined by immunoblotting. GAPDH was used as internal control. (E) Splenic Erfe mRNA levels were determined by qRT-PCR. 18S ribosomal RNA was used as an internal control (control PBS, n = 10; control AAV-HJV, n = 10; Alk2^fl/fl;Alb-Cre PBS, n = 6; Alk2^fl/fl;Alb-Cre AAV-HJV, n = 4; Alk3^fl/fl;Alb-Cre PBS, n = 4; and Alk3^fl/fl;Alb-Cre AAV-HJV, n = 5). (F) Serum sHJV protein levels were determined by immunoblotting; n = 3 per group. Significances were presented relative to the indicated control with ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001.