FIGURE 3.

Induction of HIF-1α protein by CoCl₂ treatment mimics the effect of insulin on hGH RNA levels. Immunodetection of induced HIF-1α (120 kDa) in whole cell lysate (A) and nuclear extracts (B) of primary pituitary cells treated with 250 and 500 μm CoCl₂ for 5 h is shown. GAPDH (37 kDa) and histone H1 (32 kDa) were used as loading controls for whole cell and nuclear proteins, respectively. The HIF-1α protein band (black arrowhead) and the nonspecific protein band (NS), which was only detected in whole cell lysate, are indicated. The effects of 250 and 500 μm CoCl₂ treatment on hGH (C) and mGH (D) RNA levels were assessed by qPCR after 24 h. Significant decreases in hGH but not mGH RNAs were observed. E, a ChIP assay was performed with an anti-HIF1α antibody on chromatin isolated from primary pituitary cells treated with 250 μm CoCl₂ for 24 h. Binding events were calculated and are expressed as relative mean change, as described in Fig. 2. Significant differences are indicated by ***, p < 0.001.