Specificity of interaction between TfR and TfR2.

A series of control immunoprecipitations for TfR and TfR2 was performed. Fifty micrograms of extracts from K562 cells, TRVb cells transfected with a wild-type TfR plasmid (TRVb1), TRVb cells transfected with a wild-type TfR2 plasmid (TRVb2), and a mixture of TRVb1 and TRVb2 cells were immunoprecipitated using either the TfR mouse monoclonal antibody or the TfR2 mouse monoclonal antibody and S aureus precoated with an affinity-purified rabbit antimouse antibody (Jackson Immunolabs). Immunoprecipitates and 16.7 μg of cell extracts (equivalent to one third of the amount immunoprecipitated) were run on an 8% gel under denaturing and reducing conditions, transferred to nitrocellulose, and immunodetected for TfR or TfR2 (A and B, respectively). The legend above each blot indicates the cell lines in each lane and the legend below each blot indicates the treatment of each cell line and the antibodies used for the immunoprecipitations. NS indicates cell extracts combined only with precoated S aureus; CE, cell extracts only. These results indicate that anti-TfR cannot immunoprecipitate TfR2 by itself and vice versa.