a The expression levels of Sirt1 in nuclear and cytosolic fractions were analysed in HBMVECs with exposure to NC or H/R treatments for 6 h; α-tubulin and lamin B were used as loading controls. Representative blots are presented. Bar graph indicate the level of Sirt1 in nucleus and cytosol under normoxic (NC) or hypoxia–reoxygenation (H/R) condition (n = 4, mean ± SD; *p < 0.05 versus cytosolic level of NC group by unpaired t-test). b HBMVECs were exposed to 6-h H/R, and 500 μg aliquots of cell lysates were immunoprecipitated (IP) with anti-HMGB1 antibody (2 μg/sample), and then protein binding was detected by western blotting using anti-Sirt1 and anti-HMGB1 antibodies. c, d HBMVECs (c) or Sirt1-overexpressing HBMVECs (d) were treated with H/R for 1 h and reoxygenated for the indicated times. Cell lysates (CLs) were IP with an anti-HMGB1 antibody and then acetyl-lysine (Ac-K) or HMGB1 were detected by western blot. e HBMVECs transfected with scrambled siRNA or Sirt1 siRNA were exposed to 24-h H/R. Sirt1 in CLs or extracellular HMGB1 was analysed by western blotting; β-actin was used as a loading control. Representative blots from are presented (n = 3, mean ± SD; *p < 0.05 versus H/R treated with scrambled siRNA by unpaired t-test).