Fig. 5
From: NADH elevation during chronic hypoxia leads to VHL-mediated HIF-1α degradation via SIRT1 inhibition
SIRT1 deacetylates Lys709 of HIF-1α to facilitate protein stabilization, even under conditions of protein dehydroxylation. (A) Overexpression of p300 regulated HIF-1a stability. HIF-1α protein levels were determined under hypoxic exposure for 9 h after overexpression of p300. (B) Deacetylation of HIF-1α by SIRT1. 293T cells were transiently transfected with Flag-tagged HIF-1α and HA-tagged p300 together with wt-SIRT1 (WT) or SIRT1/H363Y (DN). HIF-1α acetylation and HIF-1α-SIRT1 interaction in immunoprecipitated HIF-1α were assessed by probing with antibodies to acetylated lysine (AcK), and SIRT1, respectively. (C) 293T cells were transiently cotransfected with 2 µg of each of Flag-tagged HIF-1α, K709R or empty vector, and 2 µg of each of HA-tagged p300 and/or Myc-tagged SIRT1. (D) The stability of K709Q was compared to that of K709R and wt-HIF-1α in SIRT1-depleted HeLa cell clone either treated or not with MG132. (E) Acetylation and ubiquitination of immunoprecipitated WT-HIF-1α and K709R were assessed in in SIRT1-depleted HeLa cell clone