I286F mutation reduces the interaction of Mt2 with Hjv, ActRIIA, and Hfe. HEK293 cells were transfected individually with pCMV6-Mt2, Mt2^I286F, Mt2^S762A, Mt2^mask, pCMV9-Hjv, pCMV6-ActRIIA, or Hfe/pJB-1-B2M. Untransfected cells were used as control (Ctrl). Immunoprecipitation was performed by mixing the lysates in the presence of protease inhibitors. (A) Mt2^I286F was not coimmunoprecipitated with Hjv. A rabbit anti-HJV antibody was used for pulldown. The eluates and 10% input were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immuodetection for Hjv and Mt2 using a HRP-conjugated anti-FLAG antibody. Mt2^I286F has reduced interaction with ActRIIA (B), and both Mt2^I286F and Mt2^mask have reduced interactions with Hfe (C). Coimmunoprecipitation was performed by using a rabbit anti-MT2 antibody. An HRP-conjugated anti-FLAG antibody was used for immunodetection of ActRIIA, Hfe, Mt2, and Mt2 mutants. (D) Mt2^mask has reduced interaction with Hjv. Coimmunoprecipitation and immunodetection were performed as described in panels B and C. An HRP-conjugated anti-FLAG antibody was used for immunodetection of Hjv, Mt2, and Mt2^mask. All experiments were repeated at least 3 times, with consistent results. IB, immunoblotting; IP, immunoprecipitation.