TfR and TfR2 are associated in vitro.
Cell extracts from K562 cells were solubilized (1 × 107cells/mL) as described in “Materials and methods,” immunoprecipitated, subjected to SDS-PAGE, transferred to nitrocellulose, and immunodetected using TfR (WB:TfR) and TfR2 antibodies (WB:TfR2). (A) K562 cell extracts (50 mL) were immunoprecipitated with a sheep antihuman TfR antiserum and S aureus (IP:TfR), and detected by Western blotting using the mouse monoclonal antibody to human TfR2 (9F8 1C11) and an antimouse/HRP-conjugated secondary antibody. All blots were visualized by chemiluminescence (Pierce). Lanes are as follows: (1) K562 extract (25 mL); (2) S aureus and TfR antibody alone; (3) S aureus and K562 extract alone; and (4) S aureus,anti-TfR serum, and K562 extract. (B) K562 extract from 5 × 105 cells was immunoprecipitated with the monoclonal TfR2 antibody (9F8 1C11) and protein G–Sepharose (IP:TfR2) and detected with sheep anti-TfR serum and swine antisheep/HRP-conjugated secondary antibody on Western blots. Lanes are as follows: (1) K562 extract (25 mL); (2) protein G–Sepharose and TfR2 monoclonal antibody (9F8 1C11) only; (3) protein G–Sepharose and K562 extract (50 mL) only; and (4) protein G–Sepharose, TfR2 monoclonal antibody (9F8 1C11), and K562 extract (50 mL). (C) K562 extract (50 mL) immunoprecipitated with anti-TfR2 monoclonal antibody (9F8 1C11) (IP:TfR2) and immunodetected for TfR2 by Western blots. Lanes are as follows: (1) K562 extract (25 mL); (2) TfR2 monoclonal antibody (9F8 1C11) isolated with protein G–Sepharose; (3) K562 extract (50 mL) isolated with S aureus alone; and (4) TfR2 isolated from K562 extracts (50 mL) with anti-TfR2 monoclonal antibody (9F8 1C11) and protein G–Sepharose. (D) K562 extract (50 mL) immunoprecipitated with anti-TfR (IP:TfR) and Western blotted for TfR (WB:TfR). Lanes are as follows: (1) K562 extract (25 mL); (2) S aureus and anti-TfR serum only; (3) K562 extract (50 mL) incubated with S aureus only; and (4) K562 extract (50 mL) immunoprecipitated with anti-TfR serum and S aureus. IP indicates immunoprecipitated; WB, Western blot; and CE, cell extract.
