Substrate cleavage by Mt2^I286F. Mt2^I286F cleaves Hjv (A), Hfe (B), Alk3 (C), and ActRIIA (D) in Hep3B cells. Cotransfection of pCMV9-Hjv, pCMV6-Hfe, Alk3, or ActRIIA with pEGFP-N1 (EGFP), pCMV9-Mt2, or Mt2^I286F, aprotinin (AP) treatment, biotinylation, and immunodetection were performed as described in Figure 2A-C and E. Input and cell surface Hjv, Hfe, Alk3, ActRIIA, Mt2, and Mt2 mutants were detected by using anti-FLAG HRP, and CM Hjv was detected by using a rabbit anti-HJV antibody. All experiments were repeated at least 3 times, with consistent results. IB, immunoblotting; n.s., nonspecific band.