Biotinylated wild type or proline hydroxylated peptides

Biotinylated wild-type or proline-hydroxylated peptides (corresponding to HIF residues 556–574) were synthesized (American Peptide Company, Sunnyvale, CA, USA), dissolved in sterile water (500 μg ml^−l) and incubated with steptavidin beads (Pierce) at 4 °C for 2 h. The beads were washed twice with VHL binding buffer (20 mM Tris, pH 8, 100 mM NaCl, 1 mM EDTA, 0.5% NP40) and three times with reaction buffer (20 mM Tris, pH 7.5, 5 mM KCl, 1.5 mM MgCl₂). For each condition, 2 μg peptide 20 μl ⁻¹ beads was aliquoted into separate tubes and the reaction buffer was added, along with cofactors (100 μM 2-ketoglutaric acid, 100 μM L-ascorbic acid, 50 μM ferrous chloride). The beads and HPH cofactors were mixed at room temperature for 15 min in reaction buffer. Prior to this incubation, PA was added to the appropriate tubes. Separate in vitro translated reactions (Promega) were the source for the HPH protein and Flag-VHL protein. A 5 μl aliquot of in vitro translated HPH-2 was added to the bead-peptide mixture for 1 h at 30 °C. Subsequently, the beads were washed with VHL binding buffer and 10 μl in vitro translated Flag-VHL was added to the beads overnight at 4 °C. The beads were washed, SDS Laemmli buffer was added, the samples were boiled, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and resultant blots were probed for Flag.