FIG. 3.

Hemin-dependent decrease of IRP1_S138E expression in B6 cells. Cells expressing human wild-type IRP1, IRP1_S138A, or IRP1_S138E were treated with 100 μM desferrioxamine (DFO) or hemin for the indicated time intervals. His-tagged chimeric IRP1 was purified from cytoplasmic extracts by affinity chromatography with Ni²⁺-nitrilotriacetic acid beads. (A and B) Total lysate (A) or affinity-purified chimeric IRP1 (B) was analyzed by EMSA with a ³²P-labeled IRE probe in the absence (top) or presence (bottom) of 2% 2-mercaptoethanol (2-ME). The positions of excess free probe and specific IRP1/IRE complexes, corresponding to endogenous (end.) murine IRP1 and transfected (transf.) human His-IRP1, are indicated by arrows. (C and D) Western blotting of total lysates (C) or affinity-purified chimeric IRP1 (D) with antibodies against IRP1 and β-actin (C, bottom).