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. 2006 Jan 9;103(3):654–659. doi: 10.1073/pnas.0508423103

Fig. 3.

Fig. 3.

P317R PHD2 is defective in inhibiting HRE reporter gene activity. (A) HEK293 cells were cotransfected with 100 ng of (eHRE)3-Luc, which contains the Epo HRE, 100 ng of pRL-TK, 200 ng of either pcDNA3 or pcDNA3-HA-HIF-1α, and 0, 5, or 15 ng of pcDNA3-Flag-PHD2 (wild type or P317R). The total DNA dose was adjusted to 415 ng with pcDNA3. Eighteen hours after transfection, the cells were harvested and assayed for luciferase activity. Error bars represent standard deviations. In separate experiments, HEK293 cells were transfected with 1 μg of wild-type or P317R pcDNA3-Flag-PHD2, and 18 h later, 20-μg extracts were analyzed by Western blotting with anti-Flag or anti-β-tubulin antibodies. The positions of PHD2, β-tubulin, and a molecular weight marker are indicated. (B) HEK293 cells were cotransfected with 100 ng of (eHRE)3-Luc, 100 ng of pRL-TK, 300 ng of either pcDNA3 or pSV-Sport-HA-hHIF-2α, and 0, 2, or 6 ng of pcDNA3-Flag-PHD2 (wild type or P317R). The total DNA dose was adjusted to 506 ng with pcDNA3. Eighteen hours after transfection, the cells were harvested and assayed for luciferase activity. (C) HEK293 cells were cotransfected with 50 ng of (eHRE)3-Luc, 100 ng of pRL-TK, and either 0, 20, or 60 ng of pcDNA3-Flag-PHD2 (wild type or P317R). The total DNA dose was adjusted to 210 ng with pcDNA3. Twenty-four hours after transfection, some cells were subjected to 1% O2 for an additional 18 h. All cells were then harvested and assayed for luciferase activity. In separate experiments, HEK293 cells were transfected with 0.5 μg of wild-type or P317R pcDNA3-Flag-PHD2, and 24 h later, some cells were subjected to 1% O2 for an additional 18 h. Cellular extracts (10 μg) were analyzed by Western blotting with anti-Flag or anti-β-tubulin antibodies. (D) HEK293 cells were cotransfected with 50 ng of (eHRE)3-Luc, 100 ng of pRL-TK, and either 0 or 100 ng of wild-type or P317R pcDNA3-Flag-PHD2. The total DNA dose was equalized with pcDNA3. (Left) Cells were harvested 42 h after transfection and assayed for luciferase activity. (Right) Results from a similar experiment, except that at 24 h after transfection, cells were subjected to 1% O2 for 18 h. With all luciferase assays, activities are normalized to that of a Renilla luciferase internal transfection control. Shown are results that are representative of two or three independent experiments performed in duplicate.