Fig. 1. TfR1 and TfR2 are not involved in enhanced Tf binding and uptake in early stages of hypoxia. (A) HIF-1α expression is enhanced after exposure to low oxygen levels. Cell lysate prepared from 1x106 K562 cells, cultured for 24 hrs in hypoxic or normoxic conditions. The Western blot was probed with rabbit HIF-1 α antibody. Equal loading was confirmed by staining with monoclonal mouse ▀-actin antibody. Fold change was assayed using Scion image software. IB=immunoblotting. (B) Hypoxia treated cells express enhanced GAPDH. Control and 24 hrs hypoxia treated K562 cells were analyzed for their total GAPDH activity, p <0.05, n=2. (C) Analysis of CD71 mRNA by Real time PCR revealed ~ 4 fold increase in TfR1 mRNA, p<0.001, n=3, repeated twice. (D) K562 cells exposed to 24 hrs of hypoxia were stained for surface expression of TfR1 (CD71) and evaluated by flow cytometry. A significant reduction in cell surface expression of TfR1 as compared to control cells is evident, p<0.0001, n=104 cells for each sample, all experiments were repeated three times. (E&F) In spite of decreased TfR1 expression the total transferrin binding to cells is significantly enhanced by hypoxia. K562 and CHO-TRVb cells exposed to 24 hrs of hypoxia were assayed for transferrin binding by flow cytometry, p<0.0001, n=104. (G) Besides increase in cell surface capture, hypoxia also causes enhanced transferrin internalization by cells. Data is presented as change in mean fluorescence intensity ▒ SE as a function of time, for each time point n=104 cells. Even after 24 hrs of hypoxia cellular TfR2 levels remain unchanged: Total cell lysate (H) and membrane fraction (I) of K562 cells was prepared and run on 10% SDS PAGE, transferred to nitrocellulose membrane and probed for TfR2 using rabbit anti-human TfR2. Equal loading was confirmed by immunostaining actin (lysates) and amido black staining (membrane fractions).