Fig. 7
From: NADH elevation during chronic hypoxia leads to VHL-mediated HIF-1α degradation via SIRT1 inhibition
Chronic degradation of HIF-1α is independent of proline hydroxylation. HeLa cells were transiently transfected with plasmids encoding HA-tagged wt-HIF-1α (WT), proline mutant P402A/P564A (Mutant) and empty vector (V), or treated with DMOG (0.5mM). (A ~ C) The levels of the exogenous wt- and mutant-HIF-1α proteins (A), and the DMOG-exposed endogenous HIF-1α were determined 9 and 24 h after commencement of hypoxia (B). The effect of pyruvate on the exogenous wt- and mutant-HIF-1α proteins HIF-1α levels was examined (C). The HIF-1α hydroxylation (OH-HIF-1α) (D), and the mutant acetylation and interaction to HA-tagged VHL in HIF-1α immunoprecipitates (E) were measured in the presence of MG132 under acute and chronic hypoxia condition. (F) At the indicated times after DMOG addition, HIF-1α levels were compared between HeLa cells with SIRT1- (+) or control-siRNA transfection (-) under normoxic condition. The concentration of pyruvate and MG132 was 1 mM and 10 mM, rescpectively