rabbit monoclonal antibody against hif 1a  (Novus Biologicals)

Journal: Nutrients

Article Title: Hypoxia-Inducible Factor Inhibitors Derived from Marine Products Suppress a Murine Model of Neovascular Retinopathy

doi: 10.3390/nu12041055

Figure Lengend Snippet: Fish ingredients suppress HIFs and their target genes associated with angiogenesis in vitro. Real-time PCR was performed for hif-1a and hif-2a ( A ) and their target genes, including vegf , epo , and pdk1 , under 1% O 2 conditions in ARPE19 cells ( B ). Note that gene expression of hif-2a was suppressed by fish ingredients. Vegf , epo , and pdk1 were upregulated under 1% O 2 conditions and significantly suppressed by fish ingredients administration. Fish ingredients were added at 1 mg/mL and the hypoxic conditions were maintained for 12 h. n = 6/group. * p < 0.05, ** p < 0.01, *** p < 0.001, † p < 0.0001, ‡ p < 0.00001 compared with 1% O 2 /vehicle. Error bars indicate mean plus SD. Veh., vehicle; Topo, topotecan; epo, erythropoietin.

Article Snippet: The membranes were incubated with primary antibodies: rabbit monoclonal antibodies against HIF-1a (1:1000, Cell Signaling Technology, Danvers, MA, USA), rabbit polyclonal antibodies against HIF-2a (1:1000, NOVUS Biologicals, Centennial, CO, USA) over two nights or mouse monoclonal antibodies against b-actin (1:10,0000, Sigma-Aldrich, St Louis, MO, USA) overnight at 4°C.

Techniques: In Vitro, Real-time Polymerase Chain Reaction, Gene Expression

Journal: Nutrients

Article Title: Hypoxia-Inducible Factor Inhibitors Derived from Marine Products Suppress a Murine Model of Neovascular Retinopathy

doi: 10.3390/nu12041055

Figure Lengend Snippet: Fish ingredients suppress HIF-1a protein expression in vitro. Western blotting for HIF-1α and HIF-2α was performed under CoCl 2 condition in ARPE19 cells ( A ), and for HIF-1α under 1% O 2 condition in ARPE19 cells ( D ), under CoCl2 ( F ) or 1% O2 conditions ( H ) in 661W cells. Quantification of the blots showed that the administration of fish ingredients suppressed increased HIF-1α protein expression under CoCl 2 ( B ) or 1% O 2 conditions ( E ) in ARPE19 cells and under CoCl 2 ( G ) or 1% O 2 conditions (I) in 661W cells ( n = 3). Quantification of the blots also showed that the administration of fish ingredients suppressed the increased HIF-2α protein expression under CoCl 2 in ARPE19 cells ( C ) ( n = 3). CoCl 2 was administered at a concentration of 200 mM, fish ingredients were added at 1 mg/mL simultaneously, and cells were incubated for 24 h. The hypoxic conditions were maintained for 48 h. Note that the fish ingredients inhibited HIF-1α and HIF-2α expression induced by CoCl 2 or hypoxia. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with 1% O 2 /vehicle or CoCl 2 /vehicle. Error bars indicate mean plus SD. Veh., vehicle; Topo, topotecan; DXR, Doxorubicin.

Article Snippet: The membranes were incubated with primary antibodies: rabbit monoclonal antibodies against HIF-1a (1:1000, Cell Signaling Technology, Danvers, MA, USA), rabbit polyclonal antibodies against HIF-2a (1:1000, NOVUS Biologicals, Centennial, CO, USA) over two nights or mouse monoclonal antibodies against b-actin (1:10,0000, Sigma-Aldrich, St Louis, MO, USA) overnight at 4°C.

Techniques: Expressing, In Vitro, Western Blot, Concentration Assay, Incubation

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