IRP binding activity and ferritin protein levels in circulating monocytes. (A) Freshly isolated blood monocytes were subjected to cell-extract preparation and subsequent Western blotting for quantification of ferritin protein expression. β-actin was used as an internal control. One of 4 representative experiments is shown. In addition, intracellular ferritin concentrations of monocytes were determined by ELISA technique. The means ± SD are shown for 15 control, 9 IDA, and 13 ACD patients. Results are expressed as ng ferritin per μg total monocyte protein. *P < .001 or +P < .005 when comparing controls with ACD or IDA patients by means of Student t test, respectively; #P < .001 for comparison of ACD with IDA subjects. (B) Cellular extracts from corresponding monocytes were analyzed for IRP binding affinity by means of band shift assays. IRP binding affinity in monocytes of controls, IDA, and ACD patients is shown in the top panel. The total IRP binding capacity was determined by preincubation of extracts with 2-mercaptoethanol (+ 2-ME, bottom panel). One of 6 representative experiments is shown.