Interaction of ATF-4 with PHD3. (A) The yeast reporter strain MaV203 expressing Gal4-BD-PHD3 and Gal4-AD-ATF-4 aa 27–351 or Gal4-AD-HIF2α aa 73–870 was assayed for histidine auxotrophy. (B) His-PHD3 expressed in insect cells is captured by immobilized MBP–ATF-4. Antibodies against MBP or PHD3 were used for immunoblot detection as indicated. (C) HeLa cells were cotransfected with a firefly luciferase reporter gene plasmid driven by HREs together with a constitutively expressed renilla luciferase control vector as depicted and the expression plasmids pcDNA3.1HIF-2α, pcDNA3.1PHD3, or pcDNA3.1V5-ATF-4. After 24 hours, HeLa cells were lysed and analyzed for firefly (FL) and renilla luciferase (RL) activities. (D) HeLa cells were cotransfected with an HRE-driven firefly luciferase reporter gene plasmid and a constitutively expressed renilla luciferase control vector as in panel C and the empty control vector pcDNA3.1 or the expression plasmid pcDNA3.1V5ATF-4. Subsequently, cells were incubated at 20% O₂ or 1% O₂ for 24 hours, lysed, and analyzed for firefly and renilla luciferase activities (C,D). Shown are mean values of the FL/RL ratios (± SD) of 3 independent experiments.