Up-regulation of TfR2 protein levels is context-dependent. (A) K562 cells were incubated with or without 30 μM holoTf for 24 hours. Cell lysates containing 40 μg protein were electrophoresed on 10% SDS-polyacrylamide gels for Western blot analysis of TfR1, TfR2, ferritin, and tubulin levels as described for Figure 1C. (B) HeLa and HEK293T cells were transiently transfected with either pcDNA3.1 (mock) or pcDNA3-TfR2-FLAG. FLAG-TfR2–expressing cells were then treated with or without 30 μM holoTf for 24 hours. Western blot analysis was carried out with 25 μg HeLa cell lysates on 7% SDS-polyacrylamide gels or with 48 μg HEK293 cell lysates on 10% SDS-polyacrylamide gels. Immunoreactivity was detected using anti-TFR2 (HeLa cell lysates) or anti-FLAG (HEK293 cell lysates) antisera as described in “Materials and methods.”