Iron-dependent translational control of ferH and eALAS mRNA in LMH-2A hepatoma cells and AEV-transformed ebls (HD3E22). Cells were incubated with the iron chelator desferrioxamine (DES; 50 μmol/L) or iron-loaded transferrin (TF; 1 mg/mL iron-saturated chicken ovotransferrin) for 24 hours before harvesting. (A) Cytoplasmic extracts were separated in linear 15% to 40% sucrose gradients47 and the RNA was isolated from 18 fractions analyzed by Northern blotting. Fraction 1 corresponds to the top and fraction 18 to the bottom of the gradient. The filters were hybridized with ³²P-labeled probes specific for chicken ferH and, in case of HD3E22 ebls, subsequently with a chicken eALAS cDNA.49 The lowest panel displays the profile obtained by staining of the RNA with methylene blue (mb). The emergence of a constant molar ratio between 28S and 18S RNA (upper and lower band, respectively) around fraction 9 indicates the assembly of 80S initiation complexes and marks the boundary between the ribosome-free and polyribosome-bound compartment. Signals between gradients from the same cell type are directly comparable, because (1) the same aliquot of RNA from each fraction was blotted, (2) both filters were hybridized with the very same probe, and (3) exposed for the same time. Thus, the sum of signals over all the fractions corresponds to total cytoplasmic mRNA levels. (B) Quantitative analysis of hybridization signals from Northern blotting by laser densitometry. (Open symbols) Cells depleted of iron by Des; (solid symbols) cells under high Tf. To facilitate comparison between different experiments, the RNA content of each fraction was expressed as a percentage of the total amount of RNA contained in the gradient. (C) Ferritin protein expression in HD3E22, LMH-2A, and HeLa cells was determined by Western blotting as described in Materials and Methods. Numbers below each pair of lanes represent the factors of iron-dependent upregulation of ferritin expression (fold induction).