Figure 3.
Coimmunoprecipitation, EMSA, and transient transfection experiments. (A) Interaction of HLF or HIF1α with Arnt was investigated by coimmunoprecipitation assay using extracts from Sf9 cells transfected with baculovirus-carrying Arnt cDNA and 35S-labeled HLF or HIF1α proteins synthesized in the in vitro reticulocyte translation system as described. Lanes 1 and 5, input proteins. (B) EMSA experiment using the Epo HRE oligonucleotide. Combinations of the reaction components are indicated above the blot. (C) Transient transfection experiment in c4 cells. Combinations of effector and reporter plasmids and their amounts (μg) are indicated beneath the chart. (D) Effects of hypoxic treatment on the transcription of the reporter gene in Hep3B cells. Combinations of effector and reporter plasmids used for transfection and their amounts (μg) are indicated below the chart. Cells were grown under normoxic (N, 21% O2) and hypoxic (H, 1% O2) conditions.