Figure 2
Effects of the Pro582Ser polymorphism on HIF-1α hydroxylation. (A) GST pulldown assay. 5 μg of recombinant (His)6FlagPHD2 were incubated with 3 μg of either GST, GST-HIF-1α (531–575), or GST-HIF-1α (569–589) prebound to GSH agarose under hydroxylation conditions, as indicated. The resins were washed, and were subsequently incubated with 35S-labeled, in vitro translated HA-VHL. Bound VHL was eluted and then subjected to SDS-PAGE and autoradiography. The position of HA-VHL is indicated. "In" designates 10% of the input HA-VHL. One of two representative results is shown. (B) VBC pulldown assay. 35S labeled, in vitro translated wild type or Pro582Ser GAL4-HIF-1α (531–652) was incubated with 1 μg of (His)6-PHD2 for the indicated times. Hydroxylated reaction products were then isolated by first incubating with (His)6-Flag-VBC, and then by immunoprecipitation with anti-Flag antibodies coupled to agarose. The immunoprecipitated reaction products were subjected to SDS-PAGE and autoradiography. "In" designates 10% of the substrate. One of three representative results is shown. (C) GST pulldown assay. 1.5 μg of recombinant (His)6PHD2 was incubated with 5 μg of either GST, GST-HIF-1α (531–652), or GST-HIF-1α (531–652) Pro582Ser prebound to GSH agarose under hydroxylation conditions, as indicated. The resins were washed, and were subsequently incubated with 35S-labeled, in vitro translated FlagVHL Arg200Trp. Bound VHL was eluted and then subjected to SDS-PAGE and autoradiography. The position of VHL Arg200Trp is indicated. "In" designates 10% of the input VHL Arg200Trp. One of two representative results is shown.