Fig. 3.

Chronic hypoxia overactivates the three PHDs. (a) Autoradiogram showing PHD activity during hypoxic kinetics in HeLa cells at 1% O₂. (Inset) [³⁵S]pVHL input (I) and pVHL binding using the P402/564A mutant (DM) or the WT GST-HIF1α constructs. (b) Western blot analysis showing PHD2 and PHD3 protein levels during hypoxic kinetics. (c) To evaluate the specific contribution of each PHD isoform, 48 h before extraction, cells were transfected with siRNAs as indicated. The levels of HIF1α, HIF2α, and β-actin were analyzed by Western blotting.