Extracellular iron is not responsible for the changes in IRP1 RNA-binding activity during hypoxia. (A) RNA supershift analysis of cytosolic HEK293 cell extracts as described in Figure 1A. Complete DMEM was treated with 600-μM high-MW Df overnight to chelate iron. Cells were treated with this high-MW Df-containing medium under normoxic or hypoxic conditions for the indicated times. Experiments were performed 3 times with a representative blot shown. (B) Bar graph shows the mean percent radioactivity ± SEM, n = 3, of hypoxia-treated cell extracts relative to the normoxic control. High-MW Df-containing serum-free DMEM was treated with 55Fe-NTA overnight. Cells were labeled with 55Fe-NTA in serum-free DMEM only or with high-MW Df (600 μM)/55Fe-NTA-containing serum-free DMEM for one hour under hypoxic or normoxic conditions. The * indicates points that differ from the control with P < .03 using a Student t test.
