Figure 1.
Figure 1. Functional studies on the effect of the C42R mutation on PHD2 activity. (A) Diagram of PHD2, showing locations of zinc finger (ZF) and prolyl hydroxylase (PH) domains. Sequence of the zinc finger across various metazoan species is shown at top. Shaded residues denote zinc-chelating residues. Arrow = Cys-42; + = Tyr-41; # = His-46. Previously reported erythrocytosis-associated mutations in PHD2 reside within or near the prolyl hydroxylase domain and comprise missense (circles), nonsense (diamond), and frameshift (triangle) mutations as shown. Numbers at top and bottom indicate residue number. (B) HEK293FT cells were transfected with constructs for the indicated proteins. Constructs were prepared by standard molecular biology techniques. Cells were lysed; the Flag-tagged proteins were immunoprecipitated, and the immunoprecipitates were examined for the absence or presence of hemagglutinin (HA)-tagged PHD2 (1-196) by anti-HA western blotting. Anti-HA and anti-Flag western blots of lysates are also shown. (C) In vitro transcription and translation reactions were performed for HA-HIF-1α BAP in the absence or presence of in vitro translated Flag-tagged BirA or BirA fusion proteins, as indicated. Biotinylation of HA-HIF-1α BAP was assessed by far-western (FW) blotting using streptavidin-alkaline phosphatase conjugates. Anti-HA and anti-Flag western blots (WB) are also shown. (D) HEK293FT cells in 96-well plates were transfected with 8 ng of (eHRE)3-Luc, 8 ng of RL-TK, and either 0.8 or 2.5 ng of either pcDNA3-Flag-PHD2 or pcDNA3-Flag-PHD2 C42R. DNA doses were held constant by the addition of pcDNA3. Eight hours after transfection, cells were exposed to 1% O2 (HX) or maintained under normoxia (NX) for an additional 16 hours. All cells were lysed, and luciferase activities were measured and normalized to that of the Renilla luciferase internal transfection control. Shown are means ± standard deviation, n = 3. *P < .05 by Student t test. Anti-Flag western blot of lysates of HEK293FT cells transfected with expression constructs for WT or C42R PHD2 is also shown. (E-F left panels) HEK293FT cells were transfected with constructs for the indicated proteins. Cells were lysed; the Flag-p23 was immunoprecipitated, and the immunoprecipitates were examined for the absence or presence of HA-tagged PHD2 (1-196) by anti-HA western blotting. Anti-HA and anti-Flag western blots of lysates are also shown. (E-F right panels) HEK293FT cells in 96-well plates were transfected with 8 ng of (eHRE)3-Luc, 8 ng of RL-TK, and 2.5 ng of either pcDNA3 or the indicated PHD2 expression constructs. Eight hours after transfection, cells were exposed to 1% O2 (HX) or maintained under normoxia (NX) for an additional 16 hours. All cells were lysed, and luciferase activities were measured and normalized to that of the Renilla luciferase internal transfection control. Shown are means ± standard deviation, n = 3-4. *P < .05 by Student t test. Western blots of lysates of HEK293FT cells transfected with PHD2 expression constructs are also shown.

Functional studies on the effect of the C42R mutation on PHD2 activity. (A) Diagram of PHD2, showing locations of zinc finger (ZF) and prolyl hydroxylase (PH) domains. Sequence of the zinc finger across various metazoan species is shown at top. Shaded residues denote zinc-chelating residues. Arrow = Cys-42; + = Tyr-41; # = His-46. Previously reported erythrocytosis-associated mutations in PHD2 reside within or near the prolyl hydroxylase domain and comprise missense (circles), nonsense (diamond), and frameshift (triangle) mutations as shown. Numbers at top and bottom indicate residue number. (B) HEK293FT cells were transfected with constructs for the indicated proteins. Constructs were prepared by standard molecular biology techniques. Cells were lysed; the Flag-tagged proteins were immunoprecipitated, and the immunoprecipitates were examined for the absence or presence of hemagglutinin (HA)-tagged PHD2 (1-196) by anti-HA western blotting. Anti-HA and anti-Flag western blots of lysates are also shown. (C) In vitro transcription and translation reactions were performed for HA-HIF-1α BAP in the absence or presence of in vitro translated Flag-tagged BirA or BirA fusion proteins, as indicated. Biotinylation of HA-HIF-1α BAP was assessed by far-western (FW) blotting using streptavidin-alkaline phosphatase conjugates. Anti-HA and anti-Flag western blots (WB) are also shown. (D) HEK293FT cells in 96-well plates were transfected with 8 ng of (eHRE)3-Luc, 8 ng of RL-TK, and either 0.8 or 2.5 ng of either pcDNA3-Flag-PHD2 or pcDNA3-Flag-PHD2 C42R. DNA doses were held constant by the addition of pcDNA3. Eight hours after transfection, cells were exposed to 1% O2 (HX) or maintained under normoxia (NX) for an additional 16 hours. All cells were lysed, and luciferase activities were measured and normalized to that of the Renilla luciferase internal transfection control. Shown are means ± standard deviation, n = 3. *P < .05 by Student t test. Anti-Flag western blot of lysates of HEK293FT cells transfected with expression constructs for WT or C42R PHD2 is also shown. (E-F left panels) HEK293FT cells were transfected with constructs for the indicated proteins. Cells were lysed; the Flag-p23 was immunoprecipitated, and the immunoprecipitates were examined for the absence or presence of HA-tagged PHD2 (1-196) by anti-HA western blotting. Anti-HA and anti-Flag western blots of lysates are also shown. (E-F right panels) HEK293FT cells in 96-well plates were transfected with 8 ng of (eHRE)3-Luc, 8 ng of RL-TK, and 2.5 ng of either pcDNA3 or the indicated PHD2 expression constructs. Eight hours after transfection, cells were exposed to 1% O2 (HX) or maintained under normoxia (NX) for an additional 16 hours. All cells were lysed, and luciferase activities were measured and normalized to that of the Renilla luciferase internal transfection control. Shown are means ± standard deviation, n = 3-4. *P < .05 by Student t test. Western blots of lysates of HEK293FT cells transfected with PHD2 expression constructs are also shown.

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