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. 2015 Jan 8;290(12):7634–7646. doi: 10.1074/jbc.M114.592519

FIGURE 4.

FIGURE 4.

Catecholamines promote IRE-IRP interaction. A, RNA-EMSA was performed to assess IRE-IRP interaction in NE-treated (0–30 μm, 8 h) HepG2 cells. Cytosolic extracts (10 μg) were incubated with 32P-labeled TfR1-IRE probe, and the RNA-protein complexes were separated on non-denaturing polyacrylamide gels. Cytosolic extract from DFO (150 μm)-treated cells was used as positive control. B, 2 μg of IRP1 or IRP2 antibody (Abs) alone or together were added along with the cytosolic extracts isolated from EPI-treated HepG2 cells just prior to the addition of radiolabeled IRE probe. Similarly, IRP1 or IRP2 antibody was added in DFO-treated cytosolic extract, and gel-shift analysis was performed. Fpn antibody was added to EPI-treated cytosolic extract as nonspecific control antibody. C, RNA-EMSA was performed with cytosolic extracts from untreated, EPI/NE (0.1 μm)-, or DFO (75 μm)-treated HepG2 cells incubated with 32P-labeled Ft-IRE probe (Wild) or 32P-labeled Ft-IRE-mut probe (Mutant). D, similarly, RNA-EMSA was performed with cytosolic extracts from untreated, EPI/NE (1 μm)- or DFO (100 μm)-treated C2C12 cells incubated with 32P-labeled Ft-IRE probe or 32P-labeled Ft-IRE-mut probe. E, RNA-EMSA was performed using radiolabeled RNA probe corresponding to the 267-nucleotide Fpn 5′-UTR after incubating with cytoplasmic extracts isolated from untreated and EPI/NE (30 μm)-treated HepG2 cells. RNA-protein complexes were resolved by 5% non-denaturing polyacrylamide gels, and gels were dried and subjected to autoradiography. C, control. F, RNA-EMSA was performed using 32P-labeled Ft-IRE or 32P-labeled Ft-IRE-mut probe with cytosolic extracts isolated from livers of vehicle (V)-, EPI-, or NE-injected mice. G, similarly, RNA-EMSA was performed using 32P-labeled Ft-IRE probe with cytosolic extracts isolated from skeletal muscle of vehicle-, EPI-, or NE-injected mice. In all cases, FP denotes radiolabeled Ft-IRE probe without incubation of any cytosolic extract. Each experiment was performed at least three times independently with similar results.