Fig. 1.
Fig. 1. Oxygen-regulated ALAS2 expression. / (A) Immunoblot analysis of nuclear proteins (80 μg) prepared from undifferentiated and DMSO-treated MEL cells cultured under normoxic or hypoxic conditions. Hypoxic HeLa cells were used as a positive control. The iron chelators CPX and DFX were added where indicated, and cells were cultured under normoxic conditions. HIF-1α was detected by using a chicken anti–HIF-1 polyclonal antibody immunoglobulin Y (IgY), described previously,25 followed by a rabbit anti–chicken horseradish peroxidase–coupled secondary antibody. The signal obtained with an anti–Sp-1 antibody was used as a control for loading and transfer efficiency. (B) Representative Northern blot of undifferentiated and DMSO-treated MEL cells cultured under normoxic or hypoxic conditions. The same blot was subsequently hybridized with the indicated cDNA probes. The ribosomal protein L28 cDNA probe was used to correct for loading differences. (C) Measurement of heme pseudoperoxidase activity in undifferentiated and DMSO-treated MEL cells cultured under normoxic or hypoxic conditions for 48 hours. Heme activity was determined by colorimetry based on 2,7-diaminofluorene. Relative activity units were normalized to the normoxic pseudoperoxidase activity of undifferentiated MEL cells, which was arbitrarily defined as 1. Means ± SDs of 3 independent experiments are shown. (D) Luciferase reporter gene assays of transiently transfected HeLa cells cultured under normoxic or hypoxic conditions for 16 hours. ALAS2 promoter–driven firefly luciferase expression plasmids contained either a wild-type (pTH10) or mutant (pTH15) putative HBS. A simian virus 40 (SV40) promoter–driven luciferase reporter gene construct, containing 3 copies of either wild-type or mutant erythropoietin (EPO) 3′ enhancer–derived HBSs, served as a control. A cotransfected renilla luciferase expression vector served as an internal control for transfection efficiency and extract preparation. Relative light units were normalized to the normoxic firefly luciferase activities, which were arbitrarily defined as 1. Means ± SDs of 7 independent transfections are shown.

Oxygen-regulated ALAS2 expression.

(A) Immunoblot analysis of nuclear proteins (80 μg) prepared from undifferentiated and DMSO-treated MEL cells cultured under normoxic or hypoxic conditions. Hypoxic HeLa cells were used as a positive control. The iron chelators CPX and DFX were added where indicated, and cells were cultured under normoxic conditions. HIF-1α was detected by using a chicken anti–HIF-1 polyclonal antibody immunoglobulin Y (IgY), described previously,25 followed by a rabbit anti–chicken horseradish peroxidase–coupled secondary antibody. The signal obtained with an anti–Sp-1 antibody was used as a control for loading and transfer efficiency. (B) Representative Northern blot of undifferentiated and DMSO-treated MEL cells cultured under normoxic or hypoxic conditions. The same blot was subsequently hybridized with the indicated cDNA probes. The ribosomal protein L28 cDNA probe was used to correct for loading differences. (C) Measurement of heme pseudoperoxidase activity in undifferentiated and DMSO-treated MEL cells cultured under normoxic or hypoxic conditions for 48 hours. Heme activity was determined by colorimetry based on 2,7-diaminofluorene. Relative activity units were normalized to the normoxic pseudoperoxidase activity of undifferentiated MEL cells, which was arbitrarily defined as 1. Means ± SDs of 3 independent experiments are shown. (D) Luciferase reporter gene assays of transiently transfected HeLa cells cultured under normoxic or hypoxic conditions for 16 hours. ALAS2 promoter–driven firefly luciferase expression plasmids contained either a wild-type (pTH10) or mutant (pTH15) putative HBS. A simian virus 40 (SV40) promoter–driven luciferase reporter gene construct, containing 3 copies of either wild-type or mutant erythropoietin (EPO) 3′ enhancer–derived HBSs, served as a control. A cotransfected renilla luciferase expression vector served as an internal control for transfection efficiency and extract preparation. Relative light units were normalized to the normoxic firefly luciferase activities, which were arbitrarily defined as 1. Means ± SDs of 7 independent transfections are shown.

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