ATF-4 aa 154–181 harbors a novel ODD domain. (A) HeLa cells were transiently transfected with V5–ATF-4, V5–ATF-3, or V5–ARNT expression vectors. To control for equal transfection efficiencies, cells were cotransfected with pEGFPC1. At 6 hours after transfection, cells were exposed to 1% O2 or treated with 1 mM DMOG for 24 hours. Subsequently, cells were lysed and analyzed by immunoblotting. (B) HeLa cells were transiently transfected with different ATF-4 variants and treated as in panel A. (C) HeLa cells were transiently transfected with V5–ATF-4 or V5–ATF-4Δ aa154-181. To control for equal transfection efficiencies, cells were cotransfected with pEGFPC1. At 6 hours after transfection, cells were exposed to 1% O2. After exposure to 1% O2 for 24 hours, the cells were treated with 20 μg/mL cycloheximide and were reoxygenated with 20% O2. Cells were lysed at different reoxygenation time points, and total protein was analyzed by immunoblotting. Band intensities of 3 independent experiments were determined and half-life of the V5–ATF-4 and V5–ATF-4Δ aa154-181 proteins were calculated. (D) Decline of V5–ATF-4 and V5–ATF-4Δ aa154-181 after reoxygenation calculated from 3 independently performed experiments as described in panel C. Shown are mean values (± SD) of 3 independent experiments.
