Figure 4.

The FIH enzyme complex is the site of inhibition by peroxide. (A) Hypotonic extracts were prepared from U2OS FLAG–FIH cells treated with dox for 24 h –/+10 μM T-hydro (applied in two bolus additions 40 min apart at the end of the time course). Extracts were titrated into OH reactions with RRL GAL775–826 to enable specific assay of overexpressed FLAG–FIH. Purified FLAG–FIH (FLAG eluate) was tested in parallel. Coomassie stain of extracts and FLAG eluates (lower panel). (B) U2OS control or FLAG–FIH cells were treated with dox, either alone or in combination with 2,2′-dipyridyl (2,2′-Dip) or 0.1% O₂ and then exposed to T-hydro as in A. HIF-1α IB of extracts confirmed efficacy of the 2,2′-dipyridyl and 0.1% O₂ treatments. FLAG–FIH was then purified and FLAG eluate tested for activity. ^* Indicates a nonspecific band. Dox, doxycycline; FIH, factor inhibiting hypoxia-inducible factor; HIF, hypoxia-inducible factor; IB, immunoblotting; OH, hydroxylation; RRL, rabbit reticulocyte lysate; T-hydro, tert-butyl hydroperoxide.