Hfe interacts with Tfrc in erythroid cells and impairs Tf-bound iron uptake. (A) Immunofluorescence of MEL cells showing colocalization (yellow) of Hfe-GFP fusion protein (green) and Tfrc (red). Cells were prepared as described in supplemental Methods. Images were captured on a Nikon TE2000-U inverted microscope equipped with DIC (Nikon), a CoolSnap cf digital camera (Photometrics), and a Plan Apo objective 60×/1.40 oil (Nikon) and acquired using IP Lab Version 3.6.5a software (Scanalytics). Brightness/contrast and color balance were adjusted using Adobe Photoshop Version 7.0.1 (Adobe Systems). (B) Western blot for Tfrc after immunoprecipitation of GFP in protein lysates from MEL cells transduced with lentivirus expressing GFP or Hfe-GFP fusion protein. After stripping, the membranes were blotted for GFP, showing the expected size for GFP and Hfe-GFP in the corresponding lanes. (C) Uptake of ⁵⁹Fe by wt and Hfe-KO erythroid cells from ⁵⁹Fe-saturated Tf. Cells were harvested from phlebotomized mice and erythroid cells selected as previously described.³⁰  Counts per minute (cpm) were normalized to the percentage of Tfrc⁺ cells as detected by fluorescence-activated cell sorter (usually > 85%). Five independent experiments were performed. (D) Tfrc mean fluorescence intensity in isolated erythroid cells shows down-regulation of Tfrc in Hfe-KO cells. Twenty independent measures were made. (E) Iron uptake measured in panel C was normalized to the Tfrc mean fluorescence intensity ratio between wt and Hfe-KO cells for each experiment to control for the differences observed in Tfrc expression. Data are mean ± SEM. *P ≤ .05. ***P ≤ .001.