Figure 2. Recombinant IRP1_S711E possesses minimal aconitase and IRE-binding activities.

(A) SDS/PAGE of purified human recombinant His-tagged wild-type IRP1, IRP1_S711A and IRP1_S711E. The proteins were visualized by staining with Coomassie Brilliant Blue. (B) Purified recombinant proteins (80 ng) were analysed by EMSA with a ³²P-labelled IRE probe in the absence (upper panel) or presence (lower panel) of 2% (w/v) 2-ME. The positions of specific His–IRP1–IRE complexes and excess free probe are indicated by arrows. (C) Aconitase assay before (white bars) and after iron-sulphur cluster reconstitution with one among ferrous sulphate/cysteine (light grey bars), ferric citrate/rhodanese/thiosulphate (dark grey bars) or ferric citrate/sulphide (black bars). Values correspond to means of triplicate samples; the aconitase activity is expressed in terms of m-units/μg of recombinant protein. **P<0.01 versus untreated control (Student's t test).