The oxygen-dependent stability of ATF-4 is proline dependent. (A) HeLa cells were transiently transfected with a constitutively expressed renilla luciferase (RL) control vector, an expression plasmid containing the Gal4 AD/BD (pM3VP16) fused to ATF-4 aa 133–183; ATF-4 aa 133-183, in which prolines P156, P162, P164, P167, and P174 have been mutated to alanine; HIF-2α aa 404-568; or HIF-2α aa 404–568 P405/531A together with the Gal4-driven firefly luciferase (FL) plasmid pGRExE1bLuc. After transfection cells, were exposed to 20% or 1% O₂ for 24 hours, lysed, and analyzed for FL and RL activities. Shown are mean values plus or minus SD of the FL/RL ratios of 3 independent experiments. (B) HeLa cells were transiently transfected with V5–ATF-4, V5–ATF-4Δ aa 154-81, or V5–ATF-4 5 × P > A, in which prolines P156, P162, P164, P167, and P174 have been mutated to alanine. To control for equal transfection efficiencies cells were cotransfected with pEGFPC1. At 6 hours after transfection, cells were exposed to 20% or 1% O₂ for 24 hours. Subsequently, the cells were lysed and analyzed by immunoblotting. (C) Multiple sequence alignment of the proposed ATF-4 ODD domain.