Figure 3.

IRPs containing the functional degradation domain are ubiquitinated in iron-replete cells. RD4 stable transformants of IRP2 (A and D) or IRP1+73 (B and E) were cultured for 24 hr in the presence of dexamethasone (20 nM) to induce expression of recombinant protein. After induction, MG132 (40 μM) was added for 20 min before the addition of either 100 μg/ml FAC (F, lanes 2 and 3) or 100 μM Df (D, lanes 4 and 5) to individual plates. Lysates of the cells on each plate were prepared at 3 h (A, B, D, and E, lanes 2 and 4) or 6 h (A, B, D, and E, lanes 3 and 5) after adding Df or FAC. Alternatively, after induction of expression with dexamethasone for 24 hr, cells expressing IRP1+73 were treated with lactacystin (5 μM) and either Df (100 μM) or FAC (100 μg/ml) for 12 hr (C and F). Lysates were immunoprecipitated with anti-myc antibody, were resolved by 8% SDS/PAGE, and were transferred and probed with anti-ubiquitin (A–C), followed by stripping and reprobing of the blot with anti-myc antibody (D–F).