MT2/HJV functional interaction. (A) HeLa cells were transfected with HJV in the presence of the empty vector (mock), matriptase-2^wt (MT2^wt), MT2^G442R, MT2^D521N, MT2^E522K, MT2^G442R/D521N, MT2^D521N/E522K, and MT2^ΔSP. Whole-cell extracts (CL), concentrated media (CM), and supernatants after PI-PLC cleavage (PI-PLC) were loaded onto a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and processed for Western blot analysis. Anti-FLAG and anti-HJV were used to detect MT2 and HJV, respectively. The equal loading was verified by antitubulin. Scales refer to relative molecular mass (in kilodaltons). (B) Binding assay was used to measure m-HJV in the presence of increasing concentrations of wild-type and mutants MT2 expressing vectors and was performed essentially as described in “Functional characterization of matriptase-2 mutants.” Experiments were made in triplicate and performed 3 times. Error bars represent SD. (C) Hepcidin promoter responses by HJV, in the presence of MT2. A firefly luciferase reporter driven by 2.9 kb of the proximal hepcidin promoter was cotransfected into Hep3B cells with Renilla luciferase vector pRL-TK, either alone (HAMP) or with HJV (HAMP + HJV) combined or not with MT2-expressing vectors. Relative luciferase activity is calculated as reported in “Functional characterization of matriptase-2 mutants” and expressed as a multiple of the activity of cells transfected with the reporter alone. Experiments, made in triplicate, were performed 3 times. Error bars represent SD. (D) HeLa cells were cotransfected with wild-type and mutant matriptase-2 (MT2^WT, MT2^G442R, MT2^D521N, MT2^E522K) in the presence of HJV or of an empty vector. Precleared whole-cell extracts were immunoprecipitated with anti-HJV and revealed with the anti-FLAG antibody, which recognizes MT2. To control for transfection, whole-cell extracts (CL) were loaded and revealed with anti-HJV and/or anti-FLAG antibodies.