Intravenous AAV2/8-ALB-mHFE2-MycDDK (abbreviated as AAV-HJV) injection induces liver-specific HJV expression. Hepatocyte-specific Alk2-deficient mice (Alk2^fl/fl;Alb-Cre), hepatocyte-specific Alk3-deficient mice (Alk3^fl/fl;Alb-Cre), and their respective controls (Alk2^fl/fl or Alk3^fl/fl; ≥5 mice per group) at 8 weeks of age were intravenously injected with 5 × 10¹¹ particles of an AAV2/8 expressing Hjv-MycDDK under the control of a liver-specific promotor or PBS, and euthanized 2 weeks later. (A) Alk2 mRNA levels (control PBS, n = 10; 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) and (B) Alk3 mRNA levels in the liver were determined by qRT-PCR to verify knockdown efficiency. 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 = 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). (C) Relative hepatic Hjv mRNA levels were determined by qRT-PCR. Transcripts were normalized to 18S ribosomal RNA, and the average of control mice treated with PBS was set to 1 (control PBS, n = 14; 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 = 5; and Alk3^fl/fl;Alb-Cre AAV-HJV, n = 6). (D) HJV protein levels were determined to validate the hepatic overexpression of HJV. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as internal control. Data are presented as box plots with minimum to maximum whiskers. Significances were presented relative to the indicated control with ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001. (Alk2^fl/fl PBS, n = 3; Alk2^fl/fl;Alb-Cre PBS, n = 3; Alk2^fl/fl AAV-HJV, n = 4; Alk2^fl/fl;Alb-Cre AAV-HJV, n = 4; Alk3^fl/fl PBS, n = 3; Alk3^fl/fl;Alb-Cre PBS, n = 3; Alk3^fl/fl AAV-HJV, n = 3; Alk3^fl/fl;Alb-Cre AAV-HJV, n = 3).