Functional analysis of the IRP1/IRP2 chimeras. H1299 cells expressing wild type IRP2, IRP11–3-IRP24 or IRP21–3-IRP14 were grown for 48 h with 2 μg/ml (+) or without (-) tetracycline. (A and C) Cytoplasmic extracts were analyzed for IRE-binding activity with a 32P-labeled IRE probe in the absence or presence of 0.2 μg purified polyclonal HA antibody. The positions of the IRE/IRP complexes, the HA-supershifts and excess free IRE probe are indicated by arrows. (B) Analysis of TfR1 expression in two clones expressing IRP11–3-IRP24. Lysates were subjected to Western blotting with HA (top), TfR1 (middle) and β-actin (bottom) antibodies.