Expression of HIF-1 in human neutrophils.

(A) Ribonuclease protection assay of total RNA from monocytes (M) and neutrophils (N) was performed for EPAS-1 and HIF-1α, as detailed in “Materials and methods,” with HeLa cell RNA used as a positive control. A representative blot from 2 separate experiments is shown. (B) Neutrophils (5 × 10⁶/mL) were incubated for 3 hours in normoxic (21% oxygen) (± 1 mM DFO) or hypoxic (3% oxygen) environments, and whole-cell lysates were prepared. Hypoxia-treated HeLa cells were used as positive controls. Proteins were separated by SDS-PAGE and probed using an HIF-1α antibody (mAb 28b). Immunoreactive bands were imaged by enhanced chemiluminescence. A representative blot from 2 separate experiments is shown.