Purification of HIF PH. (a) Purification scheme. (b) Binding of ³⁵S-labeled pVHL to biotinylated HIF (556–575) peptide, which was preincubated with the indicated column fractions after TSK SP 5-PW chromatography. FT, flowthrough. (c) Conceptual ORF of EGLN1. Peptides identified by MS analysis of partially purified HIF PH are shown with single or double underline.

Inhibition of EGLN1 activity by small molecules. (a) Binding of ³⁵S-labeled pVHL to biotinylated HIF (556–575) peptide, which was preincubated with RRL in the presence of the indicated compounds (see Table 1). (b) Binding of ³⁵S-labeled pVHL to biotinylated HIF(556–575) peptide, which was preincubated with GST-EGLN1 in the presence of the indicated compounds.

Cofactor requirement for HIF PH. (a) Binding of ³⁵S-labeled pVHL in vitro translation products (IVT) to biotinylated HIF 556–575) peptide, which was preincubated with dialyzed (+) or undialyzed (−) RRL. (b and c) Binding of ³⁵S-labeled pVHL to biotinylated HIF(556–575) peptide, which was preincubated with dialyzed RRL (b) or GST-EGLN1 (c), and supplemented with the indicated cofactors.

Hydroxylation of HIF by recombinant EGLN1. (a and b) Binding of ³⁵S-labeled pVHL to biotinylated HIF(556–575) peptide, which was preincubated with EGLN1, produced in wheat germ extract (a, lane 3), or with increasing amounts of GST-EGLN1 fusion protein produced in E. coli (b, lanes 2 and 3). In parallel, peptide was preincubated with unprogrammed wheat germ extract (a, lane 2) or glutathione elution buffer (b, lane 1). (c) Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) analysis of biotinylated HIF(556–575) peptide before (Left) and after (Right) preincubation with GST-EGLN1. (d) Binding of recombinant, full-length, HIF1α (200 ng or 1 μg as indicated by triangles) to immobilized GST-pVHL, elongin B, and elongin C complexes in the absence (lanes l and 2) or presence (lanes 3 and 4) of EGLN1 produced in wheat germ extract. Bound HIF was detected by anti-HIF1α immunoblot (IB) analysis.

Stabilization of transcriptionally active HIF in cells. (a) Anti-HIF1α immunoblot analysis of the indicated cells treated overnight with 0, 10, or 25 μM FG-0041, as indicated by the triangles. (b) VEGF levels of conditioned media from comparable numbers of cells treated as in a. Error bars indicate 1 standard deviation.