Rabbit anti hif 1α antibody

The double-strand DNA probes were prepared by annealing the complementary oligonucleotides carrying the HIF-1 response element (HRE) in VEGF (II, 5′-CAGTGCATACGTGGGCTCCAACAGGTCCTC-3′) and COX-2 (5′- CAGTCTGTCCCGACGTGACTTCCTC-3′) promoters. The probes were labeled with [α-³²P]dATP. Nuclear extracts were collected, incubated with recombinant His-BTG3 (full length, N-terminal, or C-terminal domain), and then with 30–50 ng salmon-sperm DNA as well as 2 μg BSA in DNA-binding buffer containing 20 mM Hepes (pH 7.9), 2 mM MgCl₂, 0.1 mM EDTA, 10% glycerol, 2 mM spermidine, and 0.5 mM DTT for 30 min on ice. The labeled probe (2 × 10⁴ cpm) was then added to the reaction mixture and incubated for another 30 min on ice in a final volume of 20 μL. For the supershift assay, 200 ng of the rabbit-anti-HIF-1α antibody (100–134, Novus) was added. Protein–DNA complexes were separated on a 4% native polyacrylamide in 0.5x TBE at room temperature. The gels were dried before autoradiography.

Actinomycin D, carbobenzoxy-Leu-Leu-leucinal (MG132), β-mercaptoethanol and mouse anti-β-tubulin antibody were purchased from Sigma-Aldrich (St. Louis, MO, USA). Rabbit anti-Ets-1, mouse anti-GFP and rabbit anti-ubiquitin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit anti-caspase-3, rabbit anti-cleaved caspase-3 and horseradish peroxidase-conjugated anti-mouse or rabbit secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Rabbit anti-HIF-1α antibody was purchased from Novus Biologicals (Cambridge, UK). Anti-rabbit light chain secondary antibody was purchased from Chemicon (Temecula, CA, USA).