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.