FIG. 7.

Ref-1 interacts with HIF-1α in vitro. [³⁵S]methionine-labeled in vitro-translated GAL4/HIF 1-826 (A) and GAL4/HIF 531-584 (N-TAD) or GAL4/HIF 776-826 (C-TAD) (B) were incubated for 30 min at room temperature with recombinant GST/Ref-1 or GST in the presence or absence of diamide as indicated. Bound proteins were eluted in SDS sample buffer, run on SDS–7.5% (A) and 12.5% (B) polyacrylamide gels, and visualized by fluorography. Lane 6 in panel A represents 1/10 of the amount of the [³⁵S]methionine-labeled HIF-1α protein used in the binding reactions; lane 4 represents [³⁵S]methionine-labeled HIF-1α incubated with anti-GST affinity gel alone in the absence of GST/Ref-1. (C) Anti-GST Western blot. One-tenth input of proteins used in GST pull-down assays followed by elution and SDS-PAGE was analyzed by Western blotting using anti-GST antibodies. Positions of molecular mass markers are shown on the left in kilodaltons.