American Society of Hematology

Figure 2.

$Cleavage and stabilization of substrates by Mt2, the protease-dead Mt2S762A , and Mt2mask in Hep3B cells. (A) Mt2S762A but not Mt2mask increases cell surface Hjv. pCMV9-Hjv was cotransfected with an equal amount of pEGFP-N1 (EGFP), pCMV9-Mt2, Mt2S762A, or Mt2mask plasmid DNA into Hep3B cells. Approximately 30 hours’ posttransfection, fresh medium was changed with or without 10 µM aprotinin (AP). After another 24 hours of incubation, cell surface proteins were biotinylated at 4°C, followed by pull-down of the biotinylated proteins using streptavidin agarose beads. The eluted cell surface proteins, ∼10% of input lysate, and a fraction of concentrated CM were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunodetection by using anti-FLAG antibody for input and cell surface Hjv, Mt2, and Mt2 mutants or by using rabbit anti-HJV antibody for CM Hjv. Each panel was cropped from the same image. Effects of Mt2, Mt2S762A, and Mt2mask on cell surface Alk3 (B), ActRIIA (C), and Tfr2 (D). The cotransfection, biotinylation, and immunodetection were performed essentially the same as described in panel A except that Tfr2 was incubated with either AP (A) or furin convertase inhibitor (F). Alk3 and ActRIIA were subjected to immunodetection by using an anti-FLAG antibody, and Tfr2 was detected by using a rabbit anti-Tfr2 antibody. (E) Mt2S762A but not Mt2mask increases cell surface Hfe. Hep3B cells were cotransfected with pCMV6-Hfe, pJB-1-B2M, and pEGFP-N1 or pCMV6-Mt2 or Mt2S762A or Mt2mask at 1:1:1 ratios of plasmid DNA. AP treatment, the biotinylation of cell surface proteins, and immunodetection were performed as described in panel A. Hfe was detected by using an anti-FLAG antibody. (F) Diagram of N-terminal tagged mouse Hjv with the potential cleavage sites by Mt2 and furin. Quantification of cell surface Hjv band (G) and cell surface Hfe band (H) from panels A and E, respectively. All experiments were repeated at least 3 times, with consistent results. **P <.01; ***P <.001. IB, immunoblotting; n.s., nonspecific band.$

Cleavage and stabilization of substrates by Mt2, the protease-dead Mt2^S762A, and Mt2^maskin Hep3B cells. (A) Mt2^S762A but not Mt2^mask increases cell surface Hjv. pCMV9-Hjv was cotransfected with an equal amount of pEGFP-N1 (EGFP), pCMV9-Mt2, Mt2^S762A, or Mt2^mask plasmid DNA into Hep3B cells. Approximately 30 hours’ posttransfection, fresh medium was changed with or without 10 µM aprotinin (AP). After another 24 hours of incubation, cell surface proteins were biotinylated at 4°C, followed by pull-down of the biotinylated proteins using streptavidin agarose beads. The eluted cell surface proteins, ∼10% of input lysate, and a fraction of concentrated CM were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunodetection by using anti-FLAG antibody for input and cell surface Hjv, Mt2, and Mt2 mutants or by using rabbit anti-HJV antibody for CM Hjv. Each panel was cropped from the same image. Effects of Mt2, Mt2^S762A, and Mt2^mask on cell surface Alk3 (B), ActRIIA (C), and Tfr2 (D). The cotransfection, biotinylation, and immunodetection were performed essentially the same as described in panel A except that Tfr2 was incubated with either AP (A) or furin convertase inhibitor (F). Alk3 and ActRIIA were subjected to immunodetection by using an anti-FLAG antibody, and Tfr2 was detected by using a rabbit anti-Tfr2 antibody. (E) Mt2^S762A but not Mt2^mask increases cell surface Hfe. Hep3B cells were cotransfected with pCMV6-Hfe, pJB-1-B2M, and pEGFP-N1 or pCMV6-Mt2 or Mt2^S762A or Mt2^mask at 1:1:1 ratios of plasmid DNA. AP treatment, the biotinylation of cell surface proteins, and immunodetection were performed as described in panel A. Hfe was detected by using an anti-FLAG antibody. (F) Diagram of N-terminal tagged mouse Hjv with the potential cleavage sites by Mt2 and furin. Quantification of cell surface Hjv band (G) and cell surface Hfe band (H) from panels A and E, respectively. All experiments were repeated at least 3 times, with consistent results. **P <.01; ***P <.001. IB, immunoblotting; n.s., nonspecific band.

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