The HIF-1 binding site and the middle segment (CATGG) in the 3′ enhancer are not involved in the activation by RA.
(A) Whole-cell extracts (W) and nuclear extracts (N) were prepared from P19 cells cultured in 21%, 5%, and 2% oxygen for 8 hours. The extracts were subjected to Western blot analysis using an anti–HIF-1 monoclonal antibody. (B) Transient expression of Luc reporter constructs in P19 cells was assayed. After transfection, the cells were cultured for 20 hours in 21% oxygen (N) or 2% oxygen (H) in the presence and absence of 5 × 10−7 mol/L all-trans RA. Characteristics of the plasmids are shown in Figure 3 and Table 1. The inducibility by RA (the ratio of Luc activity in the presence versus absence of RA) is indicated. The inducibility of the pWT–del DR-2 construct in 21% oxygen was defined as 1. Each value is the mean ± SD of triplicate experiments.
