HFE expression in TRVb1 cells increases binding of IRP to IRE. Cells were left untreated (C) or incubated with 1 mg/mL diferric Tf (Tf), 150 μM Fe-NTA (Fe), or 100 mM desferal (Df) for 16 to 24 hours prior to harvesting. (A) Gel shift assay. Cell lysates (10 μg total protein) were incubated with approximately 50 000 cpm of 32P-labeled RNA probe, and the reaction mixtures were resolved on 7% polyacrylamide (60:1 acrylamide/bis) gels. The lysates of cells treated with desferal were also mixed with incubation buffer containing 2-mercaptoethanol to measure maximal IRP binding in each cell line (2ME). A separate lane was loaded with probe alone as a control (X). Samples containing 2ME were run at the same time on a separate gel. Gels were exposed to x-ray film for the same period of time. (B) Quantitation of the bands in panel A. The radioactivity in each band was detected and quantitated on a PhosphorImager (Amersham, Piscataway, NJ) using IP Lab Gel 1.5 software. The amount of radioactivity in each band was normalized to the total IRP binding (2ME-treated extracts of Df-treated cells) in each cell line. This experiment was repeated once for each cell line with a similar result.
