Expression of TfR and TfR2.
(A) The relative amounts of TfR2 were visualized in a variety of human cell lines by Western blots. Equal amounts of cell extracts (25 μg) were loaded onto gels and subjected to SDS-PAGE. TfR2 was detected with the mouse monoclonal antibody to human TfR2 (9F8 1C11) followed by a goat antimouse/horseradish peroxidase (HRP) secondary antibody. All blots were developed by chemiluminescence (Pierce): K562, HEL 92 (erythroid leukemia), HepG2, Huh7, Hep 3B, and SK-1 Hep (hepatoma). The TfR2 signal produces multiple bands between 97 and 105 kDa. (B) Quantitative immunoprecipitation of TfR and TfR2. K562 cells were labeled overnight with 50 μCi (1.85 MBq) 35S-methionine, lysed, and quantitatively immunoprecipitated with either a sheep antihuman TfR serum and protein A–Sepharose (Pharmacia) (TfR); cell extract and protein A–Sepharose as a control (C1); the TfR2 monoclonal antibody (9F8 1C11) and rabbit anti–mouse IgG–coated protein A–Sepharose (TfR2); or cell extract and rabbit antimouse coated protein A–Sepharose as a control (C2). Immunoprecipitated proteins were eluted with 2 × Laemmli buffer, run on an SDS-8% polyacrylamide gel, dried, and exposed to film. The relative amount of radioactivity in each band was determined by PhosphorImager analysis correcting for the Met/Cys content of each TfR. Reimmunoprecipitation of the supernatants of the immunoprecipitates showed that all of TfR and TfR2 were bound in the first immunoprecipitation (results not shown).
