FIG. 5.
(A and B) IRP1S138E, but not IRP1S138A, is sensitive to iron-mediated degradation. HEK293 cells were metabolically labeled for 2 h with [35S]methionine-cysteine and chased with cold media for the indicated time intervals in the absence of iron perturbations (control [ctrl]) or in the presence of 100 μM hemin (+hemin) or 100 μM desferrioxamine (+DFO). The half-life (t1/2) of IRP1S138A (A) or IRP1S138E (B) was assessed by quantitative immunoprecipitation of 500 μg of cytoplasmic extracts with 8.8 μg of FLAG antibody. (C and D) Hemin does not accelerate the turnover of endogenous IRP1 in HEK293 or B6 cells. HEK293 (C) or B6 cells (D) were treated as described for panels A and B, and the decay of endogenous IRP1 was assessed by quantitative immunoprecipitation of 500 μg of cytoplasmic extracts with 20 μl of IRP1 antiserum. Immunoprecipitated material was analyzed by SDS-PAGE on 8% gels, and proteins were visualized by autoradiography. Radioactive bands were quantified by phosphorimaging, and the indicated half-lives were calculated by plotting the data (data not shown).