Figure 7.

Stimulation of FBXL5 protein expression by induction of IRP1 or IRP2 or knockdown of FBXL5. A and B, HEK cells were treated with tet for 24 h to induce the expression of Myc-tagged IRP1^WT or IRP1^3C>3S. A and B, the 24-h treatment without or with tet was followed by a 4-h treatment with various combinations of 8 μm FAC, 2.5 μm MG115, and 100 μm Df. A, cell lysates were probed for IRP2, FBXL5, and Myc-tagged-IRP1 (n = 4 experiments). B, HEK cells were treated with tet to induce the expression of IRP2^FLAG for 24 h followed by a 4-h treatment with various combinations of 8 μm FAC, 2.5 μm MG115, and 100 μm Df. Cell lysates were then examined by immunoblotting using the indicated antibodies (n = 4). C, wild-type HEK 293 cells were transfected and harvested as in Fig. 1. Western blots using antibodies against FBXL5 and α-tubulin were conducted. For C, the samples used were from the same experiment as Fig. 5A, and the same α-tubulin blot is shown as a loading control for C and Fig. 5A. For all blots shown in this figure, representative blots from n = 3 experiments are shown, and tubulin was used as a loading control. Results are expressed as mean ± S.E. (error bars) for n = 3 experiments.