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Results: 9

1.
FIG. 7.

FIG. 7. From: Iron-Mediated Degradation of IRP2, an Unexpected Pathway Involving a 2-Oxoglutarate-Dependent Oxygenase Activity.

The 73-aa domain is not sufficient for iron-dependent degradation of a GFP indicator. (A) Schematic representation of GFP control and 73-aa fusion constructs. (B) Representative clones expressing each of the fusion proteins depicted schematically in panel A were treated overnight with 100 μM hemin or DFO. Lysates (30 μg) were resolved by SDS-PAGE on an 11% gel and analyzed by Western blotting with an antibody against FLAG (top) or β-actin (bottom). To visualize endogenous IRP2, additional aliquots containing 30 μg of lysates were resolved by SDS-PAGE on a 7.5% gel and analyzed by Western blotting with the IRP2 antisera (center).

Jian Wang, et al. Mol Cell Biol. 2004 February;24(3):954-965.
2.
FIG. 4.

FIG. 4. From: Iron-Mediated Degradation of IRP2, an Unexpected Pathway Involving a 2-Oxoglutarate-Dependent Oxygenase Activity.

Dose-dependent saturation of the IRP2 degradation machinery. A total of 3 × 106 H1299 cells were transiently transfected with the indicated amounts of plasmid pcDNA3-IRP2wt (A) or pcDNA3-IRP23CS (B) in 100-mm-diameter dishes and were incubated for 36 h to allow expression of the chimeric proteins. Subsequently, the cells were either left untreated (lanes 1, 4, and 7) or treated overnight with either 100 μM hemin (lanes 2, 5, and 8) or 30 μg of FAC/ml (lanes 3, 6, and 9), and lysates were analyzed by Western blotting with an anti-HA (top) or anti-β-actin (bottom) antibody.

Jian Wang, et al. Mol Cell Biol. 2004 February;24(3):954-965.
3.
FIG. 3.

FIG. 3. From: Iron-Mediated Degradation of IRP2, an Unexpected Pathway Involving a 2-Oxoglutarate-Dependent Oxygenase Activity.

The antioxidants NAC, ascorbate, and α-tocopherol promote the proteasomal degradation of wild-type IRP2 (IRP2wt) and IRP23CS. (A) Cells expressing IRP2wt were treated for 10 h with the indicated concentrations of NAC (lanes 2 to 4) or with FAC in the absence (lane 5) or presence (lane 6) of 10 mM NAC. Lysates were analyzed by Western blotting with the anti-HA (top) or anti-β-actin (bottom) antibody. (B) Cells were treated overnight with the indicated concentrations of hemin (lane 2), FAC (lane 3), or ascorbate (lanes 4 and 5), and lysates were analyzed as for panel A. (C) Cells were treated overnight with the indicated concentrations of ascorbate (lane 2) or α-tocopherol (lanes 3 to 5), and lysates were analyzed as for panel A. (D and E) Cells expressing IRP2wt (D) or IRP23CS (E) were treated for 8 h with 2.5 mM ascorbate, 20 mM NAC, or 10 mM α-tocopherol in the absence or presence of 20 μM MG132, and lysates were analyzed as for panel A.

Jian Wang, et al. Mol Cell Biol. 2004 February;24(3):954-965.
4.
FIG. 5.

FIG. 5. From: Iron-Mediated Degradation of IRP2, an Unexpected Pathway Involving a 2-Oxoglutarate-Dependent Oxygenase Activity.

Iron-dependent decrease in the expression of IRP2 mutants harboring deletions within the 73-aa domain. (A) Schematic representation of IRP2 deletion mutants. (B) Lysates from cell clones expressing wild-type IRP2 (IRP2wt) and its Δ25, Δ50, and Δ73 mutants were analyzed by Western blotting with the anti-HA antibody. The positions of molecular size standards are indicated on the right. (C, D, and E) Iron-dependent decrease in the expression of IRP2Δ25, IRP2Δ50, and IRP2Δ73, respectively. For each of the mutants depicted schematically in panel A, three representative clones expressing that mutant were treated overnight with the indicated concentrations of FAC. Lysates were analyzed by Western blotting with an anti-HA (top) or anti-β-actin (bottom) antibody. For IRP2Δ25 clones 8, 25, and 26, the ratios of chimeric IRP2 to endogenous β-actin, calculated as described in Results, were 0.022, 0.003, and 0.003, respectively. For IRP2Δ50 clones 15, 27, and 32, the respective values were 0.027, 0.003, and 0.002, and for IRP2Δ73 clones 7, 9, and 16, the respective values were 0.002, 0.004, and 0.020.

Jian Wang, et al. Mol Cell Biol. 2004 February;24(3):954-965.
5.
FIG. 9.

FIG. 9. From: Iron-Mediated Degradation of IRP2, an Unexpected Pathway Involving a 2-Oxoglutarate-Dependent Oxygenase Activity.

DMOG stabilizes IRP2 following exposure of iron-depleted cells to iron. (A and B) Pulse-chase experiment to analyze the effects of DMOG on IRP2 degradation by iron. H1299 cells expressing chimeric wild-type IRP2 (IRP2wt) were either pretreated overnight with 100 μM DFO (B) or left untreated (A). Cells were then metabolically labeled for 2 h with [35S]methionine-cysteine in the presence or absence of 5 mM DMOG. Subsequently, cells were chased for the indicated time intervals in a cold medium in the presence of 30 μg of FAC/ml. Cytoplasmic lysates were analyzed as for Fig. 2C. The radioactive bands corresponding to IRP2 (arrows) were quantified by phosphorimaging. In the graphs below the gels, the percentage of residual radioactivity from two independent experiments (mean ± standard deviation) is plotted against time. (C) Quantification of IRP2 synthesis at time zero from two independent pulse-chase experiments. Error bars, standard deviations.

Jian Wang, et al. Mol Cell Biol. 2004 February;24(3):954-965.
6.
FIG. 1.

FIG. 1. From: Iron-Mediated Degradation of IRP2, an Unexpected Pathway Involving a 2-Oxoglutarate-Dependent Oxygenase Activity.

Tetracycline-inducible expression of wild-type IRP2 (IRP2wt) or IRP23CS in H1299 cells. (A) Schematic representation of IRP2wt (top) and IRP23CS (bottom) showing domains 1 to 4, the hinge which links domains 3 and 4, and the C-terminal HA tag. The 73-aa sequence within domain 1 is shaded. The cysteines at positions 168, 174, and 178 of IRP2wt have been mutated to serines in IRP23CS. (B) H1299 cells engineered to express IRP2wt (lanes 1 to 4) or IRP23CS (lanes 5 to 8) were grown for 48 h with (+) or without (−) tetracycline at 2 μg/ml, and cytoplasmic extracts were analyzed for IRE-binding activity with a 32P-labeled IRE probe in the absence or presence of 0.2 μg of a purified polyclonal anti-HA antibody. The positions of the IRE-IRP2 complex, the HA supershift, and excess free IRE probe are indicated by arrows. (C) IRP2wt (lanes 1 to 6) or IRP23CS (lanes 7 to 12) transfectants were grown for 2, 3, or 4 days with (+) or without (−) tetracycline at 2 μg/ml, and lysates were subjected to Western blotting with antibodies against HA (top), TfR (center), and β-actin (bottom). t, time.

Jian Wang, et al. Mol Cell Biol. 2004 February;24(3):954-965.
7.
FIG. 2.

FIG. 2. From: Iron-Mediated Degradation of IRP2, an Unexpected Pathway Involving a 2-Oxoglutarate-Dependent Oxygenase Activity.

Iron-dependent turnover of wild-type IRP2 (IRP2wt) and IRP23CS. (A and B) Cells expressing IRP2wt (A) or IRP23CS (B) were treated overnight with 100 μM hemin (lanes 2 and 3) or 30 μg of FAC/ml (lanes 4 and 5) in the absence or presence of 20 μM MG132, and lysates were subjected to Western blotting with an anti-HA (top) or anti-β-actin (bottom) antibody. (C and D) Pulse-chase of IRP2wt (C) and IRP23CS (D) in the absence (top) or presence (bottom) of FAC. Control (untreated) or iron-loaded (pretreated overnight with 30 μg of FAC/ml) cells were metabolically labeled for 2 h with [35S]methionine-cysteine. Subsequently, the control or iron-loaded cells were chased for the indicated time intervals in cold medium either in the absence or in the presence of 30 μg of FAC/ml, respectively. Cytoplasmic lysates (500 μg) were subjected to quantitative immunoprecipitation with 1 μg of a purified polyclonal anti-HA antibody (Santa Cruz). Immunoprecipitated proteins were analyzed by SDS-PAGE on a 7.5% gel and visualized by autoradiography (arrows). The radioactive bands were quantified by phosphorimaging. In the graphs below the gels, the percentage of residual radioactivity from three independent experiments (mean ± standard deviation) is plotted against time.

Jian Wang, et al. Mol Cell Biol. 2004 February;24(3):954-965.
8.
FIG. 8.

FIG. 8. From: Iron-Mediated Degradation of IRP2, an Unexpected Pathway Involving a 2-Oxoglutarate-Dependent Oxygenase Activity.

Conditional inhibition of iron-mediated decrease in IRP2 expression by DMOG. (A) DMOG has no apparent iron-chelating properties. B6 and parent H1299 cells were either left untreated (lanes 1 and 4) or treated for 6 h with 5 mM DMOG (lanes 2 and 5) or 100 μM DFO (lanes 3 and 6). Cytoplasmic extracts from B6 (10 μg) or H1299 (30 μg) cells were analyzed for IRE-binding activity with a 32P-labeled IRE probe in the absence (top) or presence (bottom) of 2% 2-mercaptoethanol (2-ME). The positions of the IRE-IRP complexes and excess free IRE probe are indicated by arrows. (B) Effects of DMOG on steady-state levels of chimeric wild-type IRP2. H1299 cells were incubated in the absence of tetracycline for 2 days to express chimeric wild-type IRP2. Half of the cells were left untreated, and the other half received 100 μM DFO overnight to further stimulate IRP2 expression. Tetracycline was then added back to the medium to shut off IRP2 expression, and the cells were either left untreated or pretreated with 5 mM DMOG for 2 h. Subsequently, the cells were either left untreated (lanes 1, 2, 7, and 8) or treated in the presence or absence of 5 mM DMOG for 4 h with 30 μg of FAC/ml (lanes 3, 4, 9, and 10) or 100 μM hemin (lanes 5, 6, 11, and 12). Lysates were analyzed by Western blotting with an antibody against HA (top), HIF-1α (center), or β-actin (bottom). (C) Effects of DMOG on steady-state levels of endogenous IRP2. Parent H1299 cells were either left untreated (lanes 1 to 3) or pretreated overnight with 100 μM DFO (lanes 4 to 6). The cells were then either left untreated (lanes 1 and 4) or treated for 4 h with 30 μg of FAC/ml in the presence (lanes 3 and 6) or absence (lanes 2 and 5) of 5 mM DMOG. The inhibitor was administered 2 h prior to addition of FAC. Lysates were analyzed by Western blotting with an antibody against IRP2 (top), HIF-1α (center), or β-actin (bottom).

Jian Wang, et al. Mol Cell Biol. 2004 February;24(3):954-965.
9.
FIG. 6.

FIG. 6. From: Iron-Mediated Degradation of IRP2, an Unexpected Pathway Involving a 2-Oxoglutarate-Dependent Oxygenase Activity.

The 73-aa domain is not necessary for the iron- or antioxidant-dependent proteasomal degradation of IRP2, or for IRE-binding activity. (A) Cells expressing IRP2Δ73 were treated overnight with 30 μg of FAC/ml (lanes 2 and 3) or 100 μM hemin (lanes 4 and 5) in the absence or presence of 20 μM MG132, and lysates were subjected to Western blotting with an anti-HA (top) or anti-β-actin (bottom) antibody. (B) Pulse-chase of IRP2Δ73 in the absence (top) or in the presence (bottom) of FAC at 30 μg/ml. Experimental conditions were the same as those in Fig. 2C and D. The radioactive bands corresponding to IRP2Δ73 (arrows) were quantified by phosphorimaging. In the graph below the gel, the percentage of residual radioactivity from two independent experiments (mean ± standard deviation) is plotted against time. (C) Cells expressing IRP2Δ73 were treated for 8 h with 2.5 mM ascorbate (lanes 2 and 3), 10 mM α-tocopherol (lanes 4 and 5), or 20 mM NAC (lanes 6 and 7) in the absence or presence of 20 μM MG132, and lysates were analyzed as described for panel A. (D) Cells were grown for 48 h with (+) or without (−) tetracycline at 2 μg/ml to allow expression of IRP2Δ73, and cytoplasmic extracts were analyzed for IRE-binding activity with a 32P-labeled IRE probe in the absence (lanes 1 and 2) or presence (lanes 3 and 4) of 0.2 μg of a purified polyclonal anti-HA antibody. The positions of the IRE-IRP2Δ73 complex, the HA-supershift, and excess free IRE probe are indicated by arrows.

Jian Wang, et al. Mol Cell Biol. 2004 February;24(3):954-965.

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