Figure 3
From: Iron-dependent degradation of IRP2 requires its C-terminal region and IRP structural integrity
Pulse chase analysis of the turnover of the IRP1/IRP2 chimeras. H1299 cells expressing wild type IRP1 (A), wild type IRP2 (B), IRP11–3-IRP24 (C) or IRP21–3-IRP14 (D) were metabolically labeled for 2 h with 35S-methionine/cysteine. Subsequently, the cells were chased for the indicated time intervals in cold media in the absence or presence of 30 μg/ml FAC. Cytoplasmic lysates (500 μg) were subjected to quantitative immunoprecipitation with 1 μg HA (Santa Cruz) or FLAG (Sigma) antibodies. Immunoprecipitated proteins were analyzed by SDS-PAGE on a 7.5% gel and visualized by autoradiography. The radioactive bands were quantified by phosphorimaging. The percentage of residual radioactivity from three independent experiments (mean ± SD) is plotted against time.