Fig. 4. Effect of transiently expressed PHD fusion or FIH-1 fusion proteins on the nuclear accumulation of endogenous HIF-1 under hypoxia. PHD-EGFP fusion or FIH-1-EGFP fusion protein or control EGFP was transiently expressed in U2OS cells. The cell cultures were placed in a hypoxia workstation (1% O2) for 4 hours. Cells were then fixed in icecold methanol/acetone and immunostained for endogenous HIF-1 as detailed in Materials and Methods. The left column shows hydroxylase EGFP-fusion proteins, the middle column indirect immunofluorescence of endogenous HIF-1 and the right column (Merge) an overlay of both columns.