FIGURE 5.

Effects of IOP1 knockdown on IRP targets. A and B, HeLa cells were transfected with the indicated siRNA. On day 7, cells were also cotransfected with pIRE3-Luc and pRL-TK, and 48 h later, luciferase activities were measured (A) and total RNA extracted and measured for TfR1 mRNA levels by real-time PCR (B). In A, firefly luciferase activity was normalized to that of Renilla luciferase. In B, TfR1 mRNA levels were normalized to that of β-actin. The level of TfR1 mRNA upon IOP1 knockdown is 165% of that of control. ^*, p < 0.01. C-E, HeLa cells were transfected with the indicated siRNA. C, cytosolic extracts (20 μg) were subjected to Western blotting with the indicated antibodies. Shown is one of two to three representative experiments. D, total RNA was isolated from cells and analyzed by real-time PCR for ferritin heavy chain mRNA (means ± S.D.) and normalized to that of β-actin. Essentially the same result was obtained employing a second independent set of real-time PCR primers for ferritin heavy chain mRNA (data not shown). E, a ³²P-labeled RNA probe containing the ferritin heavy chain 5′-IRE was incubated with or without immunopurified IRP1 or IRP2 or HeLa cell cytoplasmic extract as indicated and then subjected to 5% PAGE. Arrow indicates IRP gel shift. F, cytosolic extracts (2 μg) were incubated with a ³²P-labeled RNA probe containing the ferritin heavy chain 5′-IRE and subjected to 5% PAGE. In some samples, 2% β-mercaptoethanol (βME) was included in the incubation. Arrows indicates IRP gel shift. One of three representative experiments is shown.