Figure 1.

ADI induces accelerated HIF-1α degradation via the HIF-1α polyubiquitination pathway. Western blots show that HIF-1α protein was decreased in response to ADI (a), but increased in co-treatments with CoCl₂ (150 μM) (b) or with MG-132 (10 μM) (c). Note that samples used in (a–c) were derived from the same experiment and gels/blots were processed in parallel. No increased HIF-1α degradation in response to ADI in RCC4 cells (d). (e) ADI increases ubiquitination of HIF-1α. A2058 cells transfected with HA-Ub-encoding plasmid were treated with 10 μM MG-132 in the absence or presence of ADI for 4 hr. Cell lysates were immunoprecipitated with HIF-1α antibody followed by western blotting with antibodies as indicated. (f) CoCl₂ inhibits ubiquitination of HIF-1α. A2058 cells transfected with HA-Ub-encoding plasmid were treated with 150 μM CoCl₂ in the absence or presence of ADI for 4 hr. Cell lysates were processed as above. (g) ADI enhances PHD2 enzymatic activity. A2058 cells were transfected with recombinant encoding PHD2-Flag, followed by treatment with ADI at various time points. PHD2 activity was measured from total lysates (50 μg protein) using GST-ODDD (100 ng) as a substrate. The PHD2 activity was analyzed by the production of hydroxylation at Pro-564 (HO-HIF-1α pro564) using anti-Pro564 antibody in western blot. Blottings with anti-GST and anti-Flag antibodies were used as controls for equal loading. (h) Effect of ADI on endogenous PHD2 activity using the similar procedure to (g) except no flag-PHD2 transfection was used. (i) GST-pulldown in vitro assays. Lysates from PHD2-Flag recombinant transfected A2058 cells treated with ADI at various time points were incubated with the GST-ODDD fusion proteins as indicated and GST alone (negative control), and analyzed by immunoblotting with antibodies against PHD2, Flag, and GST. (j) No effect of ADI on PHD1 activity. A2058 cells were transfected with recombinant encoding PHD1-Flag or PHD2-Flag (positive control) recombinants. The transfected cells were treated with ADI at various time points. PHD1 or PHD2 activity was measured by total lysates (50 μg) using GST-ODDD (100 ng) as a substrate.