The PHD2-p23 interaction facilitates prolyl hydroxylation of HIF-1α. A, HeLa cells were treated with control (Con) or p23 siRNA, and cellular extracts were prepared. GST or GST-HIF-1α (531–575) prebound to GSH-agarose was incubated with the extracts and washed, and bound PHD2 was then assessed by Western blotting (WB) using the indicated antibodies. B, PC3 cells were treated with the indicated concentrations of 17-AAG and then immediately subjected to normoxia (NX) or 2% O2 for 4 h. Cell lysates were prepared, and equal protein amounts were subjected to Western blotting using antibodies to the indicated proteins. C, RCC4 cells were treated with the indicated concentrations of 17-AAG and then immediately subjected to normoxia or 2% O2 for 4 or 20 h. Cell lysates were prepared, and equal protein amounts were subjected to Western blotting using antibodies to the indicated proteins. D, RCC4 cells were subjected to either 6 nm 17-AAG, 2% O2 (HX), or both for 20 h. The cells were lysed, and equal protein amounts were incubated with or without recombinant FLAG-VHL, the FLAG-VHL was immunoprecipitated, and proteins were captured by VHL analyzed by Western blotting using antibodies to HIF-1α. Lysates were also analyzed by Western blotting. E, HeLa cells were treated with the indicated siRNA and then subjected to 2% O2 for 4 h in the presence of 10 μm MG132. The cells were lysed, equal protein amounts were incubated with or without recombinant FLAG-VHL, the FLAG-VHL was immunoprecipitated, and proteins were captured by VHL analyzed by Western blotting using antibodies to HIF-1α. Lysates were also analyzed by Western blotting. F, HeLa cells were treated with the indicated siRNA and then subjected to 2% O2 for 4 h in the presence of 10 μm MG132. The cells were lysed, equal protein amounts were incubated with recombinant FLAG-VHL, the FLAG-VHL was immunoprecipitated, and proteins were captured by VHL analyzed by Western blotting using antibodies to HIF-1α. Lysates were also analyzed by Western blotting.