Journal: Journal of Biological Chemistry

Article Title: Role of Hypoxia-inducible Factor-1 in Transcriptional Activation of Ceruloplasmin by Iron Deficiency

doi: 10.1074/jbc.m000636200

Figure Lengend Snippet: FIG. 3. Binding of HIF-1 to the HRE of the Cp enhancer (by EMSA). A, Induction of complex formation by HIF-1 agonists. Hep3B cells were exposed for 8 h to 1 mM desferrioxamine (DFO), 1 mM bathophenanthroline sulfate (BPS), or 1% O2 (Hpx.). Nuclear extracts were incubated with 32P-labeled, oligonucleotide 24-mer probes con- taining either the Cp or Epo HRE. Complexes formed were resolved by 5% nondenaturing polyacrylamide gel electrophoresis and visualized by autoradiography. The positions of the putative HIF-1, constitutive (Const.), and nonspecific (NS) complexes are indicated by arrows. B, competitor binding to show specificity of HIF-1 binding to the Cp enhancer HRE. Hep3B cells were treated with 1% O2 for 9 h, and nuclear extracts were prepared as in A. A 10-, 100-, or 1000-fold molar excess of unlabeled, annealed oligonucleotide competitor representing the wild-type (wt) Cp HRE, the mutant (mut) Cp HRE, or the Epo HRE was added to the nuclear extract reaction mixture just prior to addition of radiolabeled Cp HRE probe. The mutated sequence in the Cp HRE is underlined. C, identification of HIF-1 subunits binding to the Cp en- hancer HRE by gel supershift analysis. Hep3B cells were treated with desferrioxamine, and nuclear extracts were prepared as in A. Before subjecting extracts to electrophoresis, the mixtures containing 32P- labeled Cp HRE probe was incubated with 1 ml of anti-HIF-1a, anti- HIF-1b, or both. The supershifted complex is indicated by the open- headed arrow.

Article Snippet: For gel supershift analysis, 1 ml of rabbit monoclonal antibody against HIF-1a or rabbit polyclonal antibody against ARNT/HIF-1b (both from Novus Biologicals, Littleton, CO) was added after the initial 20-min incubation, and the solution was further incubated for 30 min at 4 °C before electrophoresis.

Techniques: Binding Assay, Incubation, Labeling, Polyacrylamide Gel Electrophoresis, Autoradiography, Mutagenesis, Sequencing, Electrophoresis