Figure 3.

Assay of cellular PHD and FIH activity reveals differential and rapid inhibition of FIH by peroxide. (A) WGE or GAL28–826 WGE was incubated with cofactors (ascorbate, 2OG and Fe²⁺, +), or omissions as indicated, and extract from either control U2OS (+) or cells exposed to a single dose of 10 μM T-hydro and collected after 20 or 60 min. OH reactions were screened by IB. (B) OH reactions using Jurkat extracts from control, or cells exposed to a single dose of T-hydro 3 μM for 20 min. (C) OH reactions using Jurkat extracts from cells exposed to 10 μM T-hydro for 5 or 20 min. FIH activity was assessed by reaction with RRL GAL775–826. (D) OH reactions using Jurkat extracts from cells incubated with either T-hydro or H₂O₂ (μM, as indicated) for 20 min. FIH, factor inhibiting hypoxia-inducible factor; HIF, hypoxia-inducible factor; IB, immunoblotting; OH, hydroxylation; RRL, rabbit reticulocyte lysate; T-hydro, tert-butyl hydroperoxide; WGE, wheat germ extract; 2OG, 2-oxoglutarate.