Figure 5. The expression of N-terminally His-tagged IRP1_S711E in B6 cells is not affected by iron.

Cells expressing human wild-type IRP1, IRP1_S711A or IRP1_S711E were treated with 100 μM DFO or haemin for the indicated time intervals. His-tagged IRP1 was purified from cytoplasmic extracts by affinity chromatography with Ni-NTA beads. (A, B) Total lysates (A) or affinity-purified transfected IRP1 (B) were analysed by EMSA with a ³²P-labelled IRE probe in the absence (upper) or presence (lower panel) of 2% 2-ME. The positions of excess free probe and of specific IRP1–IRE complexes, corresponding to endogenous murine IRP1 and transfected human His–IRP1, are indicated by arrows. (C, D) Western blotting of total lysates (C) or affinity-purified transfected IRP1 (D) with antibodies against IRP1 and β-actin (lower panel in C).