TfR and TfR2 protein expression in human liver.

(A) To determine the relative levels of TfR and TfR2 protein in human liver tissue and K562 cells, 50 μg of cell extracts were Western blotted for either TfR (WB:TfR) or TfR2 (WB:TfR2). The panel on the left was developed using a mouse monoclonal antibody to human TfR2 (1:10 000), while the panel on the right was developed using a mouse monoclonal antibody to TfR (1:10 000). (B) To determine whether TfR and TfR2 interact in human liver, a series of immunoprecipitations for either TfR or TfR2, followed by immunoblotting for TfR or TfR2, was performed. Human liver extract (60 μg) was immunoprecipitated with either a sheep polyclonal antibody to TfR (1:100) and 30 μL of S aureus(IP:TfR) or with a mouse monoclonal antibody to TfR2 (1:120) and 30 μL of protein G–Sepharose (IP:TfR2), or the extract was loaded onto the gel (liver). Proteins were separated on an 8% acrylamide gel under denaturing and reducing conditions, transferred to nitrocellulose, and immunoblotted for either TfR (right panel) or TfR2 (left panel).