Effect of iron perturbations, pro- and anti-inflammatory cytokines on IRP binding affinity and TfR mRNA levels in activated macrophages.

THP-1 cells were treated with either ferric iron chloride (50 μM), desferrioxamine (DFO, 100 μM), IFN-γ (100 U/mL), LPS (10 μg/mL), a combination of IFN-γ+LPS, TNF-α (100 U/mL), or IL-10 (10 ng/mL), the latter being added to cells 2 hours prior to stimulation with the other cytokines. IRP activity was determined by electrophoreticmobility shift assays as described in “Materials and methods.” Two percent of 2-mercaptoethanol (2-ME) was used to fully activate IRP binding affinity. The resulting IRE/IRP complex is indicated by an arrow. TfR mRNA expression was carried out by Northern blot. Staining for 28 S rRNA was used to ensure that all lanes have been equally loaded. Shown is 1 of 3 experiments.