Figure 2. Daphnia Sir2 ORF produces a functional protein with catalytic activity. (A) Expression of Daphnia Sir2 in Cos-1 cells. 100 ng each of Daphnia FLAG-Sir2/pcDNA3.1-, FLAG-TRBP/pcDNA3.1-, or empty vector pcDNA3.1- were transfected in Cos-1 cells using Effectene (Qiagen) and total protein extract was prepared 24 h after transfection. Western blot analysis was performed with anti-Flag antibody (M2, Sigma). Arrows indicate Flag-Sir2 and Flag-TRBP positions. (B) Sirt1 Activity Assay. Activity assay was performed using 5 μl of in vitro translated Daphnia Sir2. Human recombinant Sirt1 was used as a positive control. Ex527, a Sirt1/Sir2 specific inhibitor was used to ensure specificity that the observed deacetylase activity was that of Daphnia Sir2. Note that Daphnia Sir2 data represented is after subtracting the background activity obtained with unprimed (no plasmid DNA added) reticulocyte lysate. Error bars indicate standard deviations from 4 replicate assays. A Student's T Test was performed to determine statistical significance. The p values are as follows: *: 5.2×10−7 (t stat: 22.2, df: 3); #: 4.8×10−7 (t stat: 22.6, df: 3).