ALK2 mutants increase hepcidin expression in vitro. (A) Hematologic parameters of the proband at 5 and 10 years of age. §Hematologic data at age 5 years are from De Falco et al.¹³  Hb, hemoglobin; MCH, mean corpuscular hemoglobin; MCV, mean corpuscular volume; PTL, platelet; RBC, red blood cell; Transf Sat, transferrin saturation; WBC, white blood cell. (B) Plasma membrane localization of wild-type ALK2 (ALK2^wt), ALK2^R206H, and ALK2^R258S was studied in HeLa cells labeled with a membrane-impermeable biotin. Cell-surface proteins were pulled down by using streptavidin beads and loaded onto a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. ALK2 was detected by using the anti-FLAG antibody. Plasma membrane protein pull down and loading were normalized using anti–pan cadherin antibody. TCL, total cell lysates. (C-D) Hep3B cells were transfected with the hepcidin promoter luciferase (Hamp-Luc) vector, thymidine kinase promoter–Renilla luciferase reporter plasmid for normalization, ALK2^wt, or empty vector and incubated with BMP6 (1 ng/mL) for 18 hours (C) or cotransfected with HJV (D). Forty-two hours after transfection, cells were lysed, and luciferase activity was measured. (E) Hep3B cells were transfected with HJV as described in panel D in the presence of ALK2^wt or ALK2^R206H or ALK2^R258S mutant. All the experiments were performed 3 times in triplicate. A representative experiment is shown for each panel. **P < .01; ***P < .001; ****P < .0001. RQ, relative quantification.