Figure 8.
Characterization of HIF-α deletion mutants. (A) Schematic of HIF-1α (left) and HIF-2α (right) deletion mutants. Full-length HIF-1α (HIF-1αTM) and HIF-2α (HIF-2αTM) proteins are normoxia-active due to the triple proline and asparagine residue mutations and contain the N-TAD, IH, and C-TAD domains. Constructs lacking the N-TAD (small or large), IH, or C-TAD were generated and the deleted amino acids are indicated. (B) HIF-α deletion proteins (labeled by star) were stable under normoxia (21% O2), and they can be detected in both C and N fractions isolated from HEK293 cells transiently transfected with the indicated plasmids under normoxia. ARNT (a nuclear protein) served as a control for the quality of cellular fractionations. (C) N-TADs of HIF-α proteins are critical in regulating HRE-dependent reporters. A WT-HRE (or Glut-1 or PGK) reporter was cotransfected with the indicated HIF expression plasmids into HEK293 cells and transfected cells cultured under normoxia were collected 24 h posttransfection and assayed for luciferase and β-Gal activities. Significant reduction of reporter gene expression was observed for HIF-α N-TAD deletion mutants, whereas deletion of IH enhanced HIF-α activity. Results are presented relative to reporter expression in HEK293 cells transfected with an empty vector.