In vitro characterization of the function of MT2 variants and evolutionary conservation of the catalytic domain. (A) Quantification of membrane-bound MT2 (MT2) by binding assay. HeLa cells were transiently transfected with the TMPRSS6 cDNA encoding MT2^736V, MT2^736A, or the empty vector (mock) and analyzed for the percentage of MT2 on the cell surface.⁶  The amount of surface MT2 was calculated as the ratio between the absorbance of unpermeabilized and permeabilized cells. Error bars indicate SD. (B) Hepcidin promoter activity assay. Hep3B cells were transiently transfected with 0.25 μg of pGL2-basic reporter vector (Promega) containing the 2.9-kb fragment of the human hepcidin promoter²³  in combination with pRL-TK Renilla luciferase vector (Promega) and HJV, as described previously.⁶  Increasing doses (from 0.002 to 0.01 μg/mL) of MT2^736V- or MT2^736A-expressing vectors were used. Relative luciferase activity was calculated as the ratio of firefly (reporter) to Renilla luciferase activity and is expressed as a multiple of the activity of cells transfected with the reporter alone. Experiments were performed in triplicate. The statistical significance is indicated above the bars. (C) Alignment of part of the serine protease domain of MT2 of different species by multiple sequence alignment ClustalW (EMBL-EBI) program. The sequence is highly conserved. The human 736 and the orthologous position in the other species are boxed.