Figure 2.

Transactivation of the 118 bp minimal luciferase reporter. (A) Sequence of the 118-bp minimal ORE used in the study with the conserved ORE depicted in boldface. (B) Effect of overexpression of each of the three proteins on the ORE reporter was analysed in transient transfection assays in 293 cells. Reporter constructs and the effector plasmids were co-transfected (1 µg each). Reporter activity (Rep) observed over control at normoxia was set to 100% in this and subsequent figures. Open bars show activity at 20% oxygen and filled bars show activity at 4% oxygen in all figures. The data represents an average of at least three experiments. (C) Representative blots for overexpression of RBPJ, CXXC5 and CHCHD2 as used in reporter assays. (D) Overexpression of HIF-1α does not affect the activity of the 118 bp-conserved luciferase reporter. The 293 cells were co-transfected with the reporter and HIF-1α and maintained at 4% O_2. Cells were analysed for reporter activity as described in the ‘Materials and Methods’ section. Data represents an average of three experiments. (E) Effect of co-overexpressing the three proteins on the ORE luciferase reporter. Cells were co-transfected with 1 µg each of RBPJ and CXXC5 (R+C5), RBPJ and CHCHD2 (R+D2) and CXXC5 and CHCHD2 (C5+D2) along with the 118-bp reporter. The data represent an average of at least three experiments (*P < 0.05; **P < 0.01).