FIG. 5.

IRP2 RNA-binding activity and TfR-1 mRNA abundance are reduced during nutrient deprivation. (A) HEK293 cells were treated overnight with 100 μM DFO and then treated with DMEM lacking both serum and glucose (deprivation) that was supplemented with 100 μM DFO for the indicated times. Cell lysates (10 μg) were used for EMSA in the presence or absence of 0.5% β-ME. IRP2 protein was supershifted with IRP2 antibody. Cell lysates (20 μg) were immunoblotted (IB) and probed sequentially with IRP2, IRP1, and actin antibodies. Quantification of EMSA activity for three independent experiments is shown. (B) HEK293 cells were treated with nonspecific (NS) or IRP1-targeted siRNA duplexes, cultured overnight in DMEM containing 10% FBS, and then treated with DMEM lacking both serum and glucose (deprivation) for 2 h. EMSA and protein analyses were performed as for panel A. (C) HEK293 cells treated as for panel B were harvested in Trizol, and TfR-1 mRNA was quantified by qRT-PCR. Quantification of four independent experiments is shown. Significance was determined on the basis of comparison to NS-siRNA-treated cells without nutrient deprivation; **, P < 0.01; ***, P < 0.0001. Error bars in panels A and C represent the standard errors of the means.