Figure 5

IRE-IRP binding activity is decreased in HF skin fibroblasts. a) Cytoplasmic extracts from non-HF and HF fibroblasts were analyzed for IRE-IRP binding activity using a gel-shift assay with a ³²P-labelled RNA probe containing a ferritin IRE sequence in the absence or presence of 2% 2-mercaptoethanol (2% 2-ME) to promote maximal IRE-IRP1 binding. Fibroblasts were cultured in the presence (+) or absence (-) of 100 μM ferric ammonium citrate (FAC) for 72 h. A lower binding activity of IRP1 was observed under basal conditions (no FAC) in HF fibroblasts. After FAC treatment, HF fibroblasts showed significantly less binding activity than controls (HF: 30 ± 7%; non-HF: 62 ± 4%). *p < 0.05.