Fig. 6

The extent of HIF-1α acetylation and HIF-1α interaction with VHL is increased during chronic hypoxia via SIRT1 inactivation. (A ~ C) HeLa cells co-transfected with Flag-tagged HIF-1α and either HA-tagged VHL (HA-VHL) were exposed to hypoxic conditions for 9 and 24 h (A and B) or 6 h (C). The effect of pyruvate (A), Myc-tagged SIRT1 (B), and either SIRT1- or control-shRNA (-) transfection (C) on HIF-1α interaction to VHL (A ~ C), acetylation, (B and C) was monitored in immunoprecipitated HIF-1α preparations, followed by probing with anti-acetyl-K and/or anti-HA antibodies, respectively. (D ~ E) The contribution of an association of VHL with HIF-1α in terms of HIF-1α stability was determined in the presence of SIRT1- (+) or control-siRNA (-), or pyruvate in RCC4 and RCC4/VHL cells. (E) The extent of acetylation of immunoprecipitated HIF-1α was assessed in RCC4 and RCC4/VHL cells treated with NAM. (F-G) Acetylation and ubiquitination of immunoprecipitated HIF-1α were assessed in RCC4/VHL cells treated with pyruvate alone (F), or with concomitant addition of NADH 6 h before harvest (G). The concentration of MG132, pyruvate and NADH was 10 mM, 1 mM, and 1 mM, respectively