Figure 2.

TfR2 and TfR1 compete for endocytosis only in the presence of holo-Tf. A) Total TfR1 levels are constant in HeLa/tTA-TfR2 cells in the presence of deferoxamine regardless of the presence of Tf or dox in the media, and TfR2 was turned off by adding dox. HeLa/tTA-TfR2 cells in the presence or absence of dox were treated with 150 µM deferoxamine for 24 hr prior to treatment with 25 µM holo-Tf or holo-bovine Tf (holo-bTf) for 30 min. Cell lysates were analyzed by immunoblot with anti-TfR2, anti-TfR1, and anti-actin antibodies. B) Lack of competition between TfR2 and TfR1 for endocytosis in absence of holo-Tf in contrast to competition of TfR1 with TfR2 in presence of holo-Tf in HeLa/tTA-TfR2 cells. HeLa/tTA-TfR2 cells in absence or presence of TfR2 were treated without or with 25 µM holo-Tf or holo-bTf for 30 min. Surface and total levels of TfR1 and TfR2 were measured by flow cytometry analysis as described in Materials and Methods. Data were evaluated by Student’s t test; p-values < 0.05 are indicated by *. C) TfR1 competes with Lamp1 for endocytosis. Surface TfR1 and Lamp1 levels were analyzed in absence of dox in HeLa/TfR1 20-2 cells by flow cytometry analysis. D) Lack of competition of TfR2 with Lamp1 for endocytosis. Surface TfR2 and Lamp1 levels were analyzed by flow cytometry analysis in the HeLa/TfR1 20-2 cells transiently transfected with TfR2 when TfR1 was turned off by the addition of dox. These experiments were repeated at least twice with similar results.