Expression of PfIRPa is iron dependent.
(A) Gel-retardation assays of human IRP1- and PfIRPa-mammalian IRE complexes are shown. Lanes 1 and 2, 32P-IRE alone; 3, 4, 11, and 12, 2-6 μg protein isolated from fructose-supplementedP falciparum cultures; 5, 6, 13, and 14, 6 μg protein isolated from cultures treated with 100 μM DFO for 18 hours; 7, 8, 15, and 16, 6 μg protein isolated from cultures supplemented with 25 μM FeCl3; 9, 10, 17, and 18, 6 μg lysate protein from a human EBV cell line expressing IRP1 and IRP2. Lanes 3 to 10, no β-mercaptoethanol; lanes 11 to 18, 1% β-mercaptoethanol was added to lysate-IRE incubation mixtures before loading the gel. The lysate proteins were incubated with 1.0 pmol/lane of 32P-IRE and resolved on an 8% polyacrylamide gel. (B) Western blot analysis of expression of P falciparum IRP. Protein (10 μg) isolated from P falciparum cultures was exposed to fructose for 18 hours (lane 1), from cultures treated with 100 μM DFO plus fructose for 18 hours (lanes 2), from cultures supplemented with 25 μM iron as FeCl3 plus fructose (lane 3), or isolated from a human EBV cell line expressing IRP1 (lane 4) were resolved on 8% SDS-polyacrylamide gel. The position of the molecular marker of 98 000 dalton is shown. The proteins were transferred to nitrocellulose and probed with antisera from nonimmune rabbits (prebleed, panel i), or with antibody 3950 (panel ii). Afterward, the nitrocellulose shown in panel B was stripped of antibody 3950 and was probed with an antibody against the plasmodial protein BiP (GRP) (panel iii). The visualization of the proteins was achieved after probing with a secondary, peroxidase-linked antibody in an enhanced chemiluminescence assay.
