Figure 1.

Synthetic growth defect in response to accumulation of the IRP1 apoprotein coupled with impaired FBXL5 expression. Cell viability was determined by trypan blue exclusion (A–F). Western blots are as described below. A and B, wild-type HEK 293 cells were transfected using either NT siRNA or siRNA targeting NuBP2 or Fam96a on days 0, 3, and 6 using an established protocol (11). For some experiments FBXL5 siRNA was then transfected into cells on day 6. Cells were then harvested, counted, and lysed on day 9. Western blots were performed for NuBP2 and tubulin. C–F, HEK 293 cells were transfected with NT or FBXL5-targeting siRNA for 48 h and then were treated without or with tet to induce IRP1^WT or IRP1^3C>3S. G, IRP2 protein level was determined in HEK cells in the absence and presence of IRP1^3C>3S, without and with FBXL5 knockdown, by immunoblotting and normalized to the level of expression of tubulin. Western blots were performed on cell lysates using the 9E10 anti-Myc monoclonal antibody to detect Myc-tagged IRP1^WT or IRP1^3C>3S or using antibodies directed against FBXL5, IRP2, or tubulin. Representative blots from n = 3 experiments are shown. Antibodies against Fam96a are not available. In B and E, some conditions included 80 μm FAC. For all panels, the 100% value represents the signal obtained from 1.0 × 10⁶ cells. Results are expressed as mean ± S.E. (error bars) for n = 3–6 separate experiments for each panel. Representative immunoblots are shown in panels A, C, D, F, and G. Immunoblot results from multiple experiments were quantified by densitometry and are shown in the accompanying graphs.