Research Paper Volume 8, Issue 2 pp 402—417

Involvement of Daphnia pulicaria Sir2 in regulating stress response and lifespan

Figure 5. D. pulicaria Sir2 activity declines with age. The deacetylase activity present in total protein extracts was assayed using a Sirt1 activity assay kit (Abcam). Daphnia extracts (1 μg total protein) prepared from the indicated ages (wk: weeks) were used (blue bars). NAD+ was added in excess (non-limiting amounts) to the assay mixtures. Recombinant human Sirt1 (Abcam) was used as a positive control (green bar; 30 ng protein). Ex527, a Sirt1/Sir2 specific inhibitor (800 nM), was utilized to ascertain that the deacetylase activity was from Sir2 and not another sirtuin (red bar for Daphnia Sir2, aged 1 week; black bar for human recombinant Sirt1, both 1 μg protein). A negative control with no extract added is represented as a gray bar. Error bars represent standard deviations. A Student T-test was performed to examine statistical significance between human Sirt1 and human Sirt1 plus Ex527: # t stat: 563.4, df: 5, p: 3.3×10−13. An ANOVA was performed (F: 911, df: 4,10, p: 9.2×10−13) followed by a post hoc Tukey test. The post hoc analysis revealed that all means were statistically different from one another for all: * (statistical significance between 1 wk and 8 wk), ** (statistical significance between 1 wk and 4 wk), *** (statistical significance between 4 wk and 8 wk), **** (statistical significance between no extract control and 1 wk) and indicate p<0.05, except between control and 1 wk plus Ex527, N.S.: not significant.